U.S. patent number 6,239,107 [Application Number 09/171,654] was granted by the patent office on 2001-05-29 for conjugates of lipophilic moieties and fragments of vasoactive intestinal peptide (vip).
This patent grant is currently assigned to Ramot University Authority for Applied Research and Industrial Development Ltd., Yeda Research & Development Co., Ltd.. Invention is credited to Matityahu Fridkin, Illana Gozes.
United States Patent |
6,239,107 |
Gozes , et al. |
May 29, 2001 |
Conjugates of lipophilic moieties and fragments of vasoactive
intestinal peptide (VIP)
Abstract
The invention concerns novel conjugates of peptide fragments of
vasoactive intestinal peptide (VIP) or analogues thereof having
3-12 amino acid residues, and lipophilic moieties, which may be
present at the N- or C-terminus. The invention further concerns
pharmaceutical compositions containing these novel conjugates which
may be used for treatment of male impotence or for the treatment of
neurodegenerative diseases.
Inventors: |
Gozes; Illana (Ramat Hasharon,
IL), Fridkin; Matityahu (Rehovot, IL) |
Assignee: |
Yeda Research & Development
Co., Ltd. (Rehovot, IL)
Ramot University Authority for Applied Research and Industrial
Development Ltd. (Tel-Aviv, IL)
|
Family
ID: |
11068793 |
Appl.
No.: |
09/171,654 |
Filed: |
April 29, 1999 |
PCT
Filed: |
April 18, 1997 |
PCT No.: |
PCT/IL97/00129 |
371
Date: |
April 29, 1999 |
102(e)
Date: |
April 29, 1999 |
PCT
Pub. No.: |
WO97/40070 |
PCT
Pub. Date: |
October 30, 1997 |
Foreign Application Priority Data
Current U.S.
Class: |
514/17.8;
514/21.7; 514/21.5; 530/327; 530/328; 530/329; 530/330; 530/331;
530/345; 514/21.8; 514/1.1; 514/21.6; 514/17.7; 514/21.9 |
Current CPC
Class: |
A61P
15/10 (20180101); C07K 14/57563 (20130101); A61P
25/28 (20180101); A61P 25/00 (20180101); A61K
38/00 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 14/575 (20060101); A61K
38/00 (20060101); A61K 038/06 (); A61K 038/07 ();
A61K 038/08 (); A61K 038/10 (); C07K 014/575 () |
Field of
Search: |
;514/12,14,15,16,17,18,21 ;530/324,327,328,329,330,331,345 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 225 020 |
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Jun 1987 |
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EP |
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0 325 044 |
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Jul 1989 |
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EP |
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0 354 992 |
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Feb 1990 |
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EP |
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0 540 969 |
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May 1993 |
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EP |
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0 620 008 |
|
Oct 1994 |
|
EP |
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Other References
Veki et al. Peptide Synthesis in Alcohol Solvents by the Mixed . .
. Bull. Chem. Soc. Japan vol. 61, No. 10, pp. 3653-3657, Oct.
1988..
|
Primary Examiner: Russel; Jeffrey E.
Attorney, Agent or Firm: Browdy and Neimark
Claims
What is claimed is:
1. A conjugate of a peptide coupled to a lipophilic moiety, wherein
the peptide has at least 3 and at most 12 amino acid residues, said
conjugate being selected from the formulae:
(i) R.sub.1 -X.sub.1 -X.sub.1 '-X.sub.1 "-X.sub.2 -NH--R.sub.2 (SEQ
ID NO:2);
(ii) R.sub.1 -X.sub.3 -Ser-X.sub.4 -Leu-Asn-NH--R.sub.2 (SEQ ID
NO:3); ##STR6##
wherein
R.sub.1 is H or a lipophilic moiety;
R.sub.2 is H, a lipophilic moiety, a lipophilic moiety substituted
by X.sub.3 -Ser-X.sub.4 -Leu-Asn-NHR.sub.1 (SEQ ID NO:79) or a
spacer consisting of 1-3 residues of a non-charged amino acid
coupled to X.sub.1 -X.sub.1 '-X.sub.1 "-X.sub.2 -NHR.sub.1 (SEQ ID
NO:80), with the proviso that at least one of R.sub.1 and R.sub.2
is a lipophilic moiety;
X.sub.1 is a covalent bond, Ala, Val, Ala-Val, Val-Ala, L-Lys,
D-Lys, Ala-Lys, Val-Lys, Ala-Val-Lys; Val-Ala-Lys or Orn;
X.sub.1 ' is L-Lys, D-Lys or Orn;
X.sub.1 " is L-Tyr, D-Tyr, Phe, Trp or the residue of p-amino
phenylalanine;
X.sub.4 is Ile or Tyr;
X.sub.5 is a residue of a hydrophobic aliphatic amino acid;
X.sub.2 is X.sub.5, X.sub.5 -Asn, X.sub.5 -Ser, X.sub.5 -Ile,
X.sub.5 -Tyr, X.sub.5 -Leu, X.sub.5 -Nle, X.sub.5 -D-Ala, X.sub.5
-Asn-Ser, X.sub.5 -Asn Ser-Ile (residues 1-4 of SEQ ID NO:75),
X.sub.5 -Asn-Ser-Tyr (residues 1-4 of SEQ ID NO:76), X.sub.5
-Asn-Ser-Ile-Leu (residues 1-5 of SEQ ID NO:75), X.sub.5
-Asn-Ser-Tyr-Leu (residues 1-5 of SEQ ID NO:76), X.sub.5
-Asn-Ser-Tyr-Leu (residues 1-5 of SEQ ID NO:76), X.sub.5
-Asn-Ser-Ile-Leu-Asn (SEQ ID NO:75) or X.sub.5 -Asn-Ser-Tyr-Leu-Asn
(SEQ ID NO:76);
X.sub.3 is a covalent bond, Asn, X.sub.5, X.sub.5 -Asn,
Tyr-X.sub.5, Tyr-X.sub.5 -Asn, Lys-X.sub.5, Lys-X.sub.5 -Asn,
Lys-Tyr-X.sub.5, Lys-Tyr-X.sub.5 -Asn (residues 4-7 of SEQ ID
NO:77), Lys-Lys-Tyr-X.sub.5 (residues 3-6 of SEQ ID NO:77),
Lys-Lys-Tyr-X.sub.5 -Asn (residues 3-7 of SEQ ID NO:77),
Val-Lys-Lys-Tyr-X.sub.5 (residues 2-6 of SEQ ID NO:78),
Val-Ala-Lys-Lys-Tyr-X.sub.5 -Asn (SEQ ID NO:77), or
Ala-Val-Lys-Lys-Tyr-X.sub.5 -Asn (SEQ ID NO:78);
X.sub.6 is a covalent bond or Asn, Ser, Ile, Tyr, Leu, Asn-Ser,
Asn-Ser-Ile, Asn-Ser-Tyr, Asn-Ser-Ile-Leu (residues 2-5 of SEQ ID
NO:75), Asn-Ser-Tyr-Leu (residues 2-5 of SEQ ID NO:76),
Asn-Ser-Ile-Leu-Asn (residues 2-6 of SEQ ID NO:75) or
Asn-Ser-Tyr-Leu-Asn (residues 2-6 of SEQ ID NO:76);
X.sub.7 is a covalent bond or Asn;
X.sub.8 is a covalent bond, X.sub.5, Tyr, Lys, Tyr-X.sub.5,
Lys-X.sub.5, Lys-Tyr-X.sub.5, Lys-Lys-Tyr-X.sub.5 (residues 3-6 of
SEQ ID NO:77), Val-Lys-Lys-Tyr-X.sub.5 (residues 2-6 of SEQ ID
NO:78), Ala-Lys-Lys-Tyr-X.sub.5 (residues 2-6 of SEQ ID NO:77), or
Ala-Val-Lys-Lys-Tyr-X.sub.5 (residues 1-6 of SEQ ID NO:78);
Z is --CONH--, NHCO--, --S--S--, --S(CH.sub.2).sub.t CO--NH-- or
--NH--CO(CH.sub.2).sub.t S--;
m is 1 or 2 when Z is --CONH--, --S--S-- or --S(CH.sub.2).sub.t
CO--NH--, or m is 2, 3 or 4 when Z is --NH--CO-- or
--NH--CO(CH.sub.2).sub.t S--;
n is 1 or 2 when Z is --NH--CO--, --S--S-- or
--NH--CO(CH.sub.2).sub.t S--, or n is 2, 3 or 4 when Z is --CONH--
or --S(CH.sub.2).sub.t CO--NH--, and
t is 1 or 2,
with the proviso that the conjugate
stearoyl-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ
ID NO:24) is excluded.
2. A conjugate according to claim 1, wherein X.sub.5 is a residue
of an D- or L-amino acid selected from Ala, Ile, Leu, Met, Val, Nva
and Nle.
3. A conjugate according to claim 1, wherein the lipophilic moiety
R.sub.1 is a saturated or unsaturated hydrocarbyl or carboxylic
acyl radical having at least 5 carbon atoms.
4. A conjugate according to claim 3, wherein R.sub.1 is selected
from: stearoyl (ST), caproyl (Cap) and lauroyl (Lau).
5. A conjugat according to claim 4, selected from the group
consisting of:
St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6);
St-Lys-Lys-Tyr-D-Ala-NH.sub.2 ;
St-Ala-Val-Lys-Lyl-Tyr-Leu-NH.sub.2 (SEQ ID NO:7);
St-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID NO:8);
St-Lys-Lys-Tyr-Leu-Nle-NH.sub.2 (SEQ ID NO:10);
St-Lys-Lys-Tyr-Val-NH.sub.2 (SEQ ID NO:9); and
St-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID NO:25).
6. A conjugate according to claim 4, selected from the group
consisting of:
St-Asn-Ser-Tyr-Leu-Asn-NH.sub.2 (SEQ ID NO:11);
St-Asn-Ser-Ile-Tyr-Asn-NH.sub.2 (SEQ ID NO:12); and
St-Lys-Lys-Tyr-Leu-Pro-Pro-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID
NO:13).
7. A conjugat according to claim 4, selected from the group
consisting of:
Lau-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:14);
Cap-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:15);
St-Lys-Tyr-Leu-NH.sub.2 ;
St-Lys-Lys-Tyr-Nle-NH.sub.2 (SEQ ID NO:16);
St-Val-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:17);
St-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID NO:18);
St-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID NO:19);
St-Lys-Lys-Tyr-Leu-Asn-NH.sub.2 (SEQ ID NO:20);
St-Lys-Lys-Tyr-Leu-Asn-Ser-NH.sub.2 (SEQ ID NO:21);
St-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-NH.sub.2 (SEQ ID NO:22); and
St-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-NH.sub.2 (SEQ ID NO:23).
8. A pharmaceutical composition comprising as an active ingredient
a conjugate according to claim 1, together with a pharmaceutically
acceptable carrier.
9. A pharmaceutical composition for the treatment of
neurodegenerative diseases comprising as an active ingredient a
conjugate selected from the group consisting of a conjugate
according to claims 5 or 7.
10. A pharmaceutical composition according to claim 9, wherein the
neurodegenerative disease is selected form the group consisting of:
Alzheimer's disease, Down Syndrome, decline in motor or cognitive
function due to ischemia, stroke, hereditary disease of the central
and peripheral nervous system, decline in motor or cognitive
function due to injury of the central or peripheral nervous system
and neuronal disorder associated with blood circulation and
neuronal survival.
11. A pharmaceutical composition according to claim 9, suitable for
nasal administration.
12. A pharmaceutical composition according to claim 11, in the form
of a nasal spray.
13. A pharmaceutical composition for the treatment of sexual
disfunctions, comprising as an active ingredient a conjugate
selected from thy group consisting of a conjugate according to
claim 6, St-Lys-Lys-Tyr-D-Ala-NH.sub.2,
St-Lys-Lys-Tyr-Leu-Nle-NH.sub.2 (SEQ ID NO: 10), and
St-Lys-Lys-Tyr-Val-NH.sub.2 (SEQ ID NO:9) together with a
pharmaceutically acceptable carrier.
14. A pharmaceutical composition according to claim 10, for the
treatment of male impotence.
15. A pharmaceutical composition according to claim 13, adapted for
transdermal application.
16. A pharmaceutical composition according to claim 15, wherein the
pharmaceutically acceptable carrier is 1-glyceryl
monocaprylate.
17. A method for treating sexual disfunction, comprising
administering a conjugate selected from the group consisting of a
conjugate according to claim 5, a conjugate according to claim 6,
and conjugate
St-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID
NO:24) to a patient in need thereof.
18. The method of claim 17, wherein the sexual disfunction is male
impotence.
19. The method of claim 17, wherein the conjugate is formulated for
transdermal administration.
20. The method of claim 17, wherein the conjugate is
St-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID
NO:24) and is combined with 1-glyceryl monocaprylate as a
carrier.
21. A method for treating a neurodegenerative disease, comprising
administering a conjugate selected from the group consisting of a
conjugate according to claim 6, St-Lys-Lys-Tyr-D-Ala-NH.sub.2,
St-Lys-Lys-Tyr-Leu-Nle-NH.sub.2 (SEQ ID NO:10), and
St-Lys-Lys-Tyr-Val-NH.sub.2 (SEQ ID NO:11) to a patient in need
thereof.
22. The method of claim 21, wherein the neurodegenerative disease
is selected from the group consisting of Alzheimer's disease, Down
Syndrome, decline in motor or cognitive function due to ischemia,
stroke, hereditary disease of the central and peripheral nervous
system, decline in motor or cognitive function due to injury of the
central or peripheral nervous system, and neuronal disorder
associated with blood circulation and neuronal survival.
23. The method of claim 21, wherein the conjugate is administered
nasally.
24. The method of claim 21, wherein the conjugate is formulated for
administration in the form of a nasal spray.
Description
FIELD OF THE INVENTION
The present invention concerns novel conjugates of a lipophilic
moiety and a peptide of 3-12 amino acids. The present invention
further concerns pharmaceutical compositions comprising as an
active ingredient said novel conjugates. The pharmaceutical
compositions of the invention are preferably used for the treatment
of male impotence or for the treatment of neurodegenerative
diseases.
BACKGROUND OF THE INVENTION
Vasoactive intestinal peptide (VIP), a 28 amino acid neuropeptide
widely distributed in the mammalian nervous system, has potent
neurotrophic actions that influence nerve cell function. In the
central nervous system, this role of VIP is translated into
developmental effects, display of growth factor activities and
maintenance of neuronal survival and function. Neurons, which are
capable of releasing VIP, innervate blood vessels throughout the
body, as well as the trachea in the lung, and the released VIP
serves as a potent vasodilator, inducing smooth muscle relaxation.
Radioligand binding assays, pharmacological experiments, molecular
cloning and development of superactive novel derivatives have
indicated several classes of VIP receptor sites and several
potential therapeutical uses.
Two possible therapeutical uses of VIP, modified VIP or lipophilic
VIP derivatives were reported in our previous Patents IL 87055, EP
0354992 and U.S. Pat. No. 5,147,855 and published patent
applications EP 0540969 and EP 0620008 which are directed to the
treatment of male impotence by transdermal administration and to
the treatment of neurodegenerative diseases, respectively.
VIP is a hydrophilic peptide of a very short half life in the serum
(Said, S. I., Editor, Vasoactive intestinal peptide in: Advances in
Peptide Hormone Research Series, Raven Press, New York, 1-512
(1982)) having the following sequence:
(SEQ ID NO:1) 1 2 3 4 5 6 7 8 9 10 11 12
His-Ser-Asp-Ala-Val-Phe-Tyr-Asp-Asn-Tyr-Thr-Arg- 13 14 15 16 17 18
19 20 21 22 23 24 Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-
25 25 27 28 Ser-Ile-Leu-Asn-NH.sub.2
To enhance its biological availability and increase its stability
the present inventors have resorted to two chemical modifications
reported in said patents and applications. The first was
lipophilization, namely, the addition of a fatty acid moiety,
designed to augment VIP's ability to penetrate biological membranes
without loss of activity; thus, stearoyl-VIP, a molecule combining
VIP with a stearic acid moiety at its N-terminal was designed (EP
0354992). The second modification was the replacement of native
amino acids with unnatural amino acids, namely, a substitution of
methionine (amino acid 17 of VIP) by norleucine, aimed at
stabilizing the molecule against oxidation as well as at increasing
lipophilicity; thus, stearoyl-Nle-VIP was designed (Gozes et al.,
Endocrinology, 134:2121-2125 (1994); Fauchere et al., Int. J.
Peptide Protein Res., 32:269-278 (1988); EP 0540969). Unmodified
VIP fragments derived from the 17-24 positions of the VIP sequence
are described in EP 0225020 as ulcer inhibitors.
A major obstacle in the use of any substance as a medicament is its
distribution in the body. The modified VIP or lipophilic VIP used
for transdermal treatment of male impotence reported in the
abovementioned EP 0354992 and EP 0540969 have to penetrate through
the dermis and reach the erectile tissues in a short a time span as
possible.
VIP, modified VIP or lipophilic VIP used to treat neurodegenerative
diseases described in EP 0620008 have to pass the blood brain
barrier in order to exert their therapeutic effect on brain
cells.
It would have been desirable, both for the purpose of treatment of
male impotence and for the purpose of administration to the CNS for
the treatment of neurodegenerative diseases, to use molecules that,
while having the physiological activity of the full VIP peptide,
are smaller in size and thus are able to improve the
bioavailability of the therapeutic compound at the target tissue.
Furthermore, smaller molecules are at times more stable to
degradation than larger molecules since, as a rule, they have less
sites available to degradation.
SUMMARY OF THE INVENTION
The present invention is based on the surprising finding that short
fragments of VIP or modified VIP conjugated to a lipophilic moiety,
which are 3-12 amino acids long, are physiologically active in the
treatment of impotence and/or neurodegenerative diseases. The
advantage of using short physiologically active peptides conjugated
to a lipophilic moiety versus the usage of the full VIP molecule is
better biodistribution and bioavailability in the body, as well as
ease of preparation. Furthermore, the invention concerns short
cyclic peptides containing said short fragments of VIP or of
modified VIP conjugated to a lipophilic moiety which in addition to
the above advantages feature the advantage of being relatively
degradation resistant.
The present invention is concerned with a conjugate of a peptide
coupled to a lipophilic moiety, wherein the peptide has at least 3
and at most 12 amino acid residues, said conjugate being selected
from the formulae:
##STR1##
wherein
R.sub.1 is H or a lipophilic moiety;
R.sub.2 is H, a lipophililc moiety, a lipophilic moiety substituted
by X.sub.3 -Ser-X.sub.4 -Leu-Asn-NHR.sub.1 (SEQ ID NO:79) or a
spacer consisting of 1-3 residues of a non-charged amino acid
coupled to X.sub.1 -X.sub.1 '-X.sub.1 "-X.sub.2 NHR.sub.1 (SEQ ID
NO:80),
with the proviso that at least one of R.sub.1 and R.sub.2 is a
lipohilic moiety;
X.sub.1 is a covalent bond, Ala, Val, Ala-Val, Val-Ala, L-Lys,
D-Lys, Ala-Lys, Val-Lys, Ala-Val-Lys; Val-Ala-Lys or Orn;
X.sub.1 is L-Lys, D-Lys or Orn;
X.sub.1 " is L-Tyr, D-Tyr, Phe, Trp or the residue of p-amino
phenylalanine;
X.sub.4 is Ile or Tyr;
X.sub.5 is a residue of a hydrophobic aliphatic amino acid;
X.sub.2 is X.sub.5, X.sub.5 -Asn, X.sub.5 -Ser, X.sub.5 -Ile,
X.sub.5 -Tyr, X.sub.5 Leu, X.sub.5 -Nle, X.sub.5 -D-Ala, X.sub.5
-Asn-Ser, X.sub.5 -Asn-Ser-Ile (residue 1-4 of SEQ ID NO:75),
X.sub.5 -Asn-Ser-Tyr (residues 1-4 of SEQ ID NO:76); X.sub.5
-Asn-Ser-Ile-Leu (residues 1-5 of SEQ ID NO:75), X.sub.5
-Asn-Ser-Tyr-Leu (residues 1.5 of SEQ ID NO:76), X.sub.5
-Asn-Ser-Ile-Leu-Asn (SEQ ID NO:75) or X.sub.5 -Asn-Ser-Tyr-Leu-Asn
(SEQ ID NO:76);
X.sub.3 is a covalent bond, Asn, X.sub.5, X.sub.5 -Asn,
Tyr-X.sub.5, Tyr-X.sub.5 -Asn, Lys-X.sub.5, Lys-X.sub.5 -Asn,
Lys-Tyr-X.sub.5, Lys-Tyr-X.sub.5 -Asn (residues 4-7 of SEQ ID
NO:77), Lys-Lys-Tyr-X.sub.5 ((residues 3-6 of SEQ ID NO:77),
Lys-Lys-Tyr-X.sub.5 -Asn (residues 3-7 of SEQ ID NO:77),
Val-Lys-Lys-Tyr-X.sub.5 (residues 2-6 SEQ ID NO:78),
Val-Ala-Lys-Lys-Tyr-X.sub.5 -Asn (SEQ ID NO:77), or
Ala-Val-Lys-Lys-tyr-X.sub.5 -Asn (SEQ ID NO:78);
X.sub.6 is a covalent bond or Asn, Ser, Ile, Tyr, Leu, Asn-Ser,
Asn-Ser-Ile, Asn-Ser-Tyr, Asn-Ser-Ile-Leu (residues 2-5 of SEQ ID
NO:75), Asn-Ser-Tyr-Leu (residues 2-5 of SEQ ID NO:76),
Asn-Ser-Ile-Leu-Asn (residues 2-6 of SEQ ID NO:75) or
Asn-Ser-Tyr-Leu-Asn (residues 2-6 of SEQ ID NO:76);
X.sub.7 is a covalent bond or Asn;
X.sub.8 is a covalent bond, X.sub.5, Tyr, Lys, Tyr-X.sub.5,
Lys-X.sub.5, Lys-Tyr-X.sub.5, Lys-Lys-Tyr-X.sub.5 (residues 3-6 of
SEQ ID NO:77), Val-Lys-Lys-Tyr-X.sub.5 (residues 2-6 of SEQ ID
NO:78), Ala-Lys-Lys-Tyr-X.sub.5 (residues 2-6 of SEQ ID NO:77), or
Ala-Val-Lys-Lys-Tyr-X.sub.5 (residues 1-6 of SEQ ID NO:78);
Z is --CCNH--, --NHCO--, --S--S--, --S(CH.sub.2).sub.t CO--NH-- or
--NH--CO(CH.sub.2).sub.t S--;
m is 1 or 2 when Z is --CONH--, --S--S-- or --S(CH.sub.2).sub.t
CO--NH--, or m is 2, 3 or 4 when Z is --NH--CO-- or
--NH--CO(CH.sub.2).sub.t S--;
n is 1 or 2 when Z is --NH--CO--, --S--S-- or
--NH--CO(CH.sub.2).sub.t S--, or n is 2, 3 or 4 when Z is --CONH--
or --S(CH.sub.2).sub.t CO--CO--NH--, and
t is 1 or 2,
with the proviso that the conjugate
stearoyl-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ
ID NO:24) is excluded.
The hyrdophobic aliphatic amino acid represented above by X.sub.5
may be a residue of D- or L- amino acid selected from Ala, Ile,
Leu, Met, Val, Nva and Nle.
The term "lipophilic moiety of the conjugates of the invention"
will refer in the following description and claims to: a saturated
or unsaturated hydrocarbyl or carboxylic acyl radical having at
least 3 carbon atoms such as propionyl, caproyl, laurly, palmitoyl,
stearoyl, oleyl, eicosanoyl, docsanoyl and the respective
hydrocarbyl radicals propyl, hexyl, dodecyl, hexadecyl, octadecyl,
eicosanyl and docosanyl. Preferably the hydrocarbyl or acyl radical
is saturated, and has 3-22 carbon atoms.
The term "spacer" refers to residue of a non-charged natural or
non-natural amino acid such as alanine, proline and aminocaproic
acid.
Examples of the conjugates of the invention are conjungates of a
lipophilic moiety and peptides of the sequence Lys-Lys-Tyr-Leu
derived from position 20-23 of the VIP sequence (SEQ ID NO:1)
and/or peptides of the sequence Asn-Ser-Ile-Leu-Asn, derived from
positions 24-28 of the VIP sequence (SEQ ID NO:1). modified
peptides thereof in which amino acid residues have been replaced,
added, deleted or chemically modified or combinations of these two
sequences such as:
St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6);
St-Lys-Lys-Tyr-D-Ala-NH.sub.2 ;
St-Ala-Val-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:7);
St-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID NO:8);
St-Lys-Lys-Tyr-Val-NH.sub.2 (SEQ ID NO:9);
St-Ser-Ile-Lau-Asn-NH.sub.2 ;
St-Lys-Lys-Tyr-Leu-Nle-NH.sub.2 (SEQ ID NO:10);
St-Asn-Ser-Tyr-Leu-Asn-NH.sub.2 (SEQ ID NO:11);
St-Asn-Ser-Ile-Tyr-Asn-NH.sub.2 (SEQ ID NO:12);
St-Lys-Lys-Tyr-Leu-Pro-Pro-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID
NO:13;
Lau-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:14);
Cap-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:15);
St-Lys-Tyr-Leu-NH.sub.2 ;
St-Lys-Lys-Tyr-Nle-NH.sub.2 (SEQ ID NO:16);
St-Val-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:17);
St-Leu-Asn-Ser-Ile-Leu-Asu-NH.sub.2 (SEQ ID NO:18;
St-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID NO:19);
St-Lys-Lys-Tyr-Leu-Asn-NH.sub.2 (SEQ ID NO:20);
St-Lys-Lys-Tyr-Leu-Asn-Ser-NH.sub.2 (SEQ ID NO:21);
St-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-NH.sub.2 (SEQ ID NO:22); and
St-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-NH.sub.2 (SEQ ID NO:23).
In the following, the symbol "St" stands for stearoyl, "Lau" stands
for lauroyl and "Cap" stands for caproyl.
By another aspect the present invention concerns pharmaceutical
compositions comprising as an active ingredient, active conjugates
of the invention together with a pharmaceutically acceptable
carrier.
The pharmaceutical composition of the invention comprising a
conjugate of the invention or the conjugate
St-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID
NO:24) (hereinafter "Peptide") described in EP 0620008 may be used
for the treatment of sexual disfunctions such as male impotence,
preferably by transdermal or urinary tract application. Preferred
conjugates used for this purpose are:
St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6);
St-Lys-Lys-Tyr-D-Ala-NH.sub.2 ;
St-Ala-Val-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:7);
St-Asn-Ser-Ile-Leu-Asn-NH.sub.2 ;
St-Lys-Lys-Tyr-Val-NH.sub.2 (SEQ ID NO:9);
St-Ser-Ile-Lau-Asn-NH.sub.2 ;
St-Asn-Ser-Tyr-Leu-Asn-NH.sub.2 (SEQ ID NO:11);
St-Asn-Ser-Ile-Tyr-Asn-NH.sub.2 ;
St-Lys-Lys-Tyr-Leu-Nle-NH.sub.2 (SEQ ID NO:10); and
St-Lys-Lys-Tyr-Leu-Pro-Pro-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID
NO:13).
The pharmaceutical compositions of the invention may also be used
for the treatment of neurodegenerative diseases, such as Alzheimer,
Down Syndrome, hypoxia, decline in motor or cognitive function due
to ischemia, stroke, hereditary diseases of the central and
peripheral nervous system, decline in motor or cognitive function
due to injury of the central or peripheral nervous system, decline
in cognitive functions due to old age and neurological disorders
associated with blood circulation and neuronal survival. The term
"treatment" should be understood in the context of the present
invention as alleviation, improvement or abolishment of the
abnormal conditions manifested in those diseases and more
particularly to improvement in cognitive functions damaged by those
diseases. Preferred conjugates used for this purpose are:
St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6);
St-Lys-Lys-Tyr-D-Ala-NH.sub.2 ;
St-Ala-Val-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:7);
St-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID NO:8);
St-Lys-Lys-Tyr-Val-NH.sub.2 (SEQ ID NO:9);
St-Ser-Ile-Lau-Asn-NH.sub.2 ;
Lau-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:14);
Cap-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:15);
St-Lys-Tyr-Leu-NH.sub.2 ;
St-Lys-Tyr-Leu-NH.sub.2 ;
St-Lys-Lys-Tyr-Nle-NH.sub.2 (SEQ ID NO:16);
St-Val-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:17);
St-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID NO:18);
St-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID NO:19);
St-Lys-Lys-Tyr-Leu-Nle-NH.sub.2 (SEQ ID NO:10);
St-Lys-Lys-Tyr-Leu-Asn-NH.sub.2 (SEQ ID NO:20);
St-Lys-Lys-Tyr-Leu-Asn-Ser-NH.sub.2 (SEQ ID NO:21);
St-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-NH.sub.2 (SEQ ID NO:22); and
St-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-NH.sub.2 (SEQ ID NO:23).
Optional modes of administration of pharmaceutical compositions of
the invention are subcutaneous, intravenous, oral, nasal, ocular,
by an intracerebroventricular pump, through the urinary tract or
transdermal administration.
Where the pharmaceutical compositions of the invention are used to
treat impotence, urinary tract or transdermal administration are
preferable. For transdermal application the carrier is preferably
selected from amongst those which enhance the tissue penetration of
the active ingredient. Examples of suitable carriers are olive oil,
glycerine, lubricants, nitroglycerin and Sefso.TM., and mixtures
thereof. Sefsol is a trademark (Nikko Chemicals, Tokyo) for,
1-glyceryl monocaprylate, propylene glycol didecanoate, propylene
glycol dicaprylate, glyceryl tricaprylate and sorbitan
monocaprylate and they are the preferred carriers in compositions
according to the invention. Of these, 1-glyceryl monocaprylate and
olive oil are particularly preferred. For urinary tract application
a gel is preferably used as a carrier.
The present invention further provides, for the sustained release
of a conjugate of the invention, a transdermal dispenser comprising
an applicator loaded with said conjugate and adapted for
application to the skin.
If desired, the conjugate in the applicator may be formulated into
a pharmaceutical composition of the kind specified above.
Treating male impotence by transdermal administration exhibits
several advantages over modes of parenteral, such as subcutaneous,
administration. For one, it is non-surgical and does not entail
tissue destruction. Moreover, it does not cause priapism or the
burning pain associated with other modes of administration.
Furthermore, the transdermal application is a much more discreet
and convenient mode of application as compared to an
intracavernosal injection. Transdermal administration enables the
use of a continuous slow release device which may enable
spontaneous sexual activity without the need for a lengthy
preparation, thus sparing an inflicted individual much of the usual
embarrassment.
Where the pharmaceutical compositions of the invention are to be
used as drugs acting on the central nervous system, it is
preferable to administer them through the nose, which enables the
penetration of the aerosol composition to the CNS through the
olfactory nerve (WO 91/07947), via the ocular route (Chiou, G. C.
Y., (1991) An. Rev. PharmacoL Toxical., 31:457-67) or by any other
suitable method of administration as described in W. M. Pardridge,
Peptide Drug Delivery, Raven Press, N.Y. 1991.
The pharmaceutical compositions of the invention may be also
directly targeted to the brain by an intracerebroventricular
pump.
The present invention further concerns a method of treatment of
neurodegenerative diseases or male impotence by administering to a
host in need of such treatment a therapeutically effective amount
of the conjugate of the invention.
The present invention still further provides use of the conjugate
of the invention for the preparation of a pharmaceutical
composition.
As will be appreciated by any person versed in the art, the
conjugates as defined above in formulae I above include a large
number of possible conjugates. Those which fall under the scope of
the invention and those defined as "active conjugates" in the
pharmraceutical composition of the invention are the conjugates
which are active in at least one of the following assays:
(1) conjugates which are able to induce erection in an animal model
of impotence (normal and castrated animals);
(2) conjugates which have the activity of protecting electrically
blocked neurons from death;
(3) conjugates which are able to protect untreated neurons in
culture from naturally occurring death;
(4) conjugates which are able to protect cultured neurons from
death caused by a 25-35 fragment of .beta.-amyloid peptide;
(5) conjugates which are able to avoid deterioration of learning
and memory acquisition of either old animals or animals treated
with a dementia causing agent as tested in an acceptable learning
of memory acquisition assay, as well as conjugates which are able
to improve recollection of a previously acquired task in animals
treated with a dementia causing agent, for example, as described in
example.
(6) conjugates which are able to avoid or ameliorate decline of
motor and cognitive functions in an animal model of ischemia and/or
models of stroke;
(7) conjugates which are able to protect neurons from damage caused
due to lack of oxygen;
(8) conjugates which are able to improve motor and cognitive
functions in models for hereditary neurodegenerative diseases of
the central and peripheral nervous system such as models of mice
with a knock out of ApoE (Cell, 71:343 (1992)); transgenic models
of amyloid over-expression (Nature, 373:523 (1995)); models for ALS
which are mutant super oxide dismutase expression (Science, 264,
1772 (1994)) and a model for Down Syndrome which is trisomy of
chromosome 16; and
(9) conjugates which are able to improve motor and cognitive
functions in models of injury of the central and peripheral nervous
system such as lesions of the nucleus basalis in rats (PNAS,
85:9481 (1988)); scopolamine-induced acetylcholine release in
ventral hippocampus (PNAS, 90:11287 (1993)); NMDA induced
convulsions (Brain Res. 448:115 (1988)).
In the following, the invention will be further illustrated with
reference to some non-limiting drawings and examples.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the effect of varying concentrations of
St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6) on the survival of
neurons treated with .beta.-amyloid peptide;
FIG. 2 shows the effect of the cholinergic blocker AF64A
(.largecircle.) and of AF64A together with
St-Lys-Lys-Tyr-Leu-NH.sub.2 SEQ ID NO:6) (.circle-solid.) on
learning and memory in an animal model of Alzheimer;
FIG. 3 shows the effect of the cholinergic blocker AF64A (e) and of
AF64A together with St-Lys-Lys-Tyr-D-Ala-NH.sub.2
(.tangle-solidup.) on learning and memory in an animal model of
Alzheimer;
FIG. 4 shows the effect of administration of saline control
(.largecircle.); saline together with St-Lys-Lys-Tyr-D-Ala-NH.sub.2
(.box-solid.); the cholinergic blocker AF64A (.tangle-solidup.) and
AF64A together with St-Lys-Lys-Tyr-D-Ala-NH.sub.2 (.diamond-solid.)
on memory retention in an animal model of Alzheimer;
FIG. 5 shows motor function of animals treated with saline
(.circle-solid.); saline and St-Lys-Lys-Tyr-D-Ala-H.sub.2
(.box-solid.); the cholinergic blocker AF64A (.tangle-solidup.) and
animals treated with AF64A together with
St-Lys-Lys-Tyr-D-Ala-NH.sub.2 (.diamond-solid.);
FIG. 6 shows the effect of administration of saline (.largecircle.)
and the conjugate St-Lys-Lys-Tyr-D-Ala (.quadrature.) on learning
and memory in normal control animals administered with the
vehicle;
FIG. 7 shows the effect of administration of saline
(.circle-solid.); saline and St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID
NO:6) (.box-solid.); the cholinergic blocker AF64A
(.tangle-solidup.) and AF64A together with
St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6) (.diamond-solid.) on
memory retention of a previously learned task in an animal model of
Alzheimer (first swim);
FIG. 8 shows the same experiment as described in connection with
FIG. 7 for the second swim including administration of saline and
St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6); and of AF64A together
with St-Lys-Try-Leu-NH.sub.2 (SEQ ID NO:6);
FIG. 9 shows the same experiment as described in connection with
FIG. 7 measuring the time spent by the animals in the area where
the platform used to be;
FIG. 10 shows the effect of St-Lys-Lys-Tyr-Leu-NH.sub.2
(St-KKYL-NH.sub.2 (SEQ ID NO:6) and saline on forelimb placing
behavior acquisition in normal newborn mice
(St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6) (.tangle-solidup.);
saline (.largecircle.)), and on ApoE deficient newborn mice (APO
E); (St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6) (.tangle-solidup.);
saline (.circle-solid.)).
FIG. 11 shows for comparison the effect of St-Nle.sup.17
-VIP-NH.sub.2 and saline on forelimb placing behavior acquisition
in normal newborn mice (St-Nle.sup.17 -VIP-NH.sub.2
(.tangle-soliddn.); saline (.circle-solid.), and on ApoE deficient
newborn mice (St-Nle.sup.17 -VIP-NH.sub.2 (.tangle-solidup.);
saline (.diamond-solid.));
FIG. 12 shows the effect of St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID
NO:6) (St-KKYL-NH.sub.2) and saline on cliff avoidance acquisition
in normal newborn mice (St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6),
(.tangle-soliddn.); saline. (.circle-solid.)); and ApoE deficient
mice (St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6) (.tangle-solidup.);
saline (.diamond-solid.));
FIG. 13 shows for comparison the effect of St-Nle.sup.17
-VIP-NH.sub.2 on cliff avoidance acquisition in normal newborn mice
and ApoE deficient mice;
FIG. 14 shows the Choline Acetyl transfcrasc activity of control,
and Apo-E deficient mice treated with St-Lys-Lys-Tyr-Leu-NH.sub.2
(SEQ ID NO:6);
FIG. 15 shows the HPLC analysis of brain extracts of rats
intranasally administered with .sup.125
I-St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6);
FIG. 16 shows the effect of St-Ala-Val-Lys-
Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID NO:24) (peptide 6)
in DMSO on the number of E2 and cups;
FIG. 17 shows the effect of
St-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID
NO:24) (peptide 6) and of St-Ala-Val-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ
ID NO:7) (peptide 26) in DMSO on the number of cups and on the
number of E2;
FIG. 18 shows the effect of St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID
NO:6) in 5% Sefsol.TM./20% isopropanol on the number of cups and on
the number of E2 (C=vehicle control; E=experimental peptide);
FIG. 19 shows the effect of St-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID
NO:25) in Sefsol.TM./isopropanol on the number of cups and on the
number of E2 (C=vehicle control; E=experimental peptide);
FIG. 20 shows the effect of St-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID
NO:8) in Sefsol.TM./isopropanol on the number of cups and on the
number of E2 (C=vehicle control; E=experimental peptide);
FIG. 21 shows the effect of St-Asn-Ser-Ile-Tyr-Asn-NH.sub.2 (SEQ ID
NO:22) in Sefsol.TM./isopropanol on the number of cups and on the
number of E2 (C=vehicle control; E experimental peptide);
FIG. 22 shows the effect of St-Asn-Ser-Tyr-Leu-Asn-NH.sub.2 (SEQ ID
NO:11) in Sefsol.TM./isopropanol on the number of cups and on the
number of E2;
FIG. 23 shows the effect of St-Lys-Lys-Tyr-D-Ala-NH.sub.2 (KKYO)
and St-Lys-Lys-Tyr-Val-NH.sub.2 (KKYV) (SEQ ID NO:9) on the number
of cups;
FIG. 24 shows the effect of St-Lys-Lys-Tyr-D-Ala-NH.sub.2 (KKYO)
and St-Lys-Lys-Tyr-Val-NH.sub.2 (KKYV) (SEQ ID NO:9) on the latency
to the first cup;
FIG. 25 shows the effect of St-Lys-Lys-Tyr-D-Ala-NH.sub.2 (KKYO)
and St-Lys-Lys-Tyr-Val-NH.sub.2 (KKYV) (SEQ ID NO:9) on the number
of E2;
FIG. 26 shows the effect of St-Lys-Lys-Tyr-Leu-Nle-NH.sub.2 (SEQ ID
NO:10) on the number of cups;
FIG. 27 shows the effect of
St-Lys-Lys-Tyr-Leu-Pro-Pro-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID
NO:13) on the number of cups;
FIG. 28 shows biodistribution following topical administration of
.sup.125 I-St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6);
FIG. 29 shows the HPLC analysis of intestinal extract of animals
topically administered with .sup.125 I-St-Lys-Lys-Tyr-Leu-NH.sub.2
(St-KKYL-NH.sub.2) (SEQ ID NO:6); and
FIG. 30 shows the effect of injection into the corpus cavernosum to
vehicle, 0.1 .mu.g of St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6),
and 10 .mu.g St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6) on penile
blood pressure.
DETAILED DESCRIPTION OF THE INVENTION
A. Synthesis of Linear Peptides
To obtain a large battery of small peptides an automatic peptide
synthesizer was utilized. Syntheses of the peptides of the
invention were achieved by automatic procedure employing an ABIMED
AMS 422 synthesizer (ABIMED, Langenfeld, Germany) using the
commercially available protocols via the Fmoc strategy. All
protected amino acid derivatives were as recommended by the
company. Thus, the following side-chain protection was utilized:
Lys, N-epsilon-t-butyloxycarbonyl (Boc), Tyr, Thr, Ser, O-t-butyl;
Arg; 2,2,5,7,8-pentamethylchroman-6-sulfonyl (PMC); Trp, N.sup.int
-Boc; Cys, S-trityl, Asn, beta-trityl, PyBOP, i.e.
benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate, was used as a coupling agent. Peptide chains
were assembled on a 4-([2",4"-dimethoxyphenyl] Fmoc aminoethyl)
phenoxy resin (Rink amide Resin, Nova, Switzerland).
Final cleavage of the peptide chain from the resin along with the
side chain deprotection was achieved as follows: cleavage mixture:
90% TFA, 5% water, 5% triethylsilane. The resin, 100 mg, loaded
with peptide was incubated for 30 min with a 3 ml cleavage mixture
inside the reaction column used for solid phase synthesis. After 30
min, the reaction was separated from the cleaved resin and cleavage
continued for an additional 3 hrs. The cleaved peptide was
precipitated with ice cold tert-butylmethyl ether and centrifuged
(4.degree. C., 2000 rpm). To ensure optimal precipitation,
petroleum ether (b.p. 40-60.degree. C., 1:1 v/v) was occasionally
added. The solution was decanted and the pellet was dissolved in
water and frozen for lipophilization to yield a white powder.
Purification of the crude peptides was performed by
semi-preparative HPLC on an RP-8 column (Merck 7 .mu.M;
250.times.10 mm) employing linear gradient established between 35%
acetonitrile in water containing 0.1% TFA, and 0.1% TFA in 75%
acetonitrile in water at a flow rate of 10 m/min. Elution was
monitored at 220 nm. Yields were 30-45%. Purity of the products was
ascertained by analytical HPLC on an RP-18 column (Merck;
250.times.4 mm) and amino acid analysis following exhaustive acid
hydrolysis gave the expected values of each constituent amino
acid.
Examples of the conjugates comprising linear peptides that were
synthesized by this method are those listed on pages 5-9
hereinbefore and the following conjugates are:
SEQ. SEQ. ID. ID. NO: NO: 1. CapKKYLZZ* 26 29. StYLNSILN* 19 2.
LauKKYLZ* 27 30. StKKYLNle* 10 3. KKYLZ* 28 31. StKKYLO* 49 4.
KKYLZZ* 29 32. StKKYLL* 50 5. KKYLB* 30 33. StKBYL* 51 6. KKYLZZZ*
31 34. StBKYL* 52 7. LauNSILNZ* 32 35. StBBYL* 53 8. NSILNZ* 33 36.
StKKFL* 54 9. NSILNZZ* 34 37. StKKOL* 10. CapNSILNZZ* 35 38.
StKKYZ* 55 11. NSILNZZZ* 36 39. StKKWL* 56 12. NSILNB* 37 40.
StKKXL* 57 13. KKYLZNSILN* 38 41. StKOrnYl* 58 14. KKYLZZNSILN* 39
42. StOrnKYL* 59 15. KKYLZZZNSILN* 40 43. StOrnOrnYL* 60 16.
KKYLBNSILN* 41 44. OIKKYL* 61 17. StKKYLXXXNSILN* 42 45.
PropylKKYL* 62 18. StKKLYAAANSlLN* 43 46. StKKYLAAKKYL* 63 19.
StKKYLPNSILN* 44 47. StKKYLPPKKYL* 64 20. StKKYLPPNSILN* 13 48.
StKKYLAcaKKYL* 65 21. StKYLNSILN* 45 49. StKKYLAm.Lauryl 66 KKYL*
22. StKKYLNSILN* 46 50. StKKYLNle* 10 23. StKKYLN* 20 51. StKKYLdA*
24. StKKYLNS* 21 52. StKKYLL* 67 25. StKKYLNSI* 22 53. CapKKYL* 15
26. StKKYLNSIL* 23 54. LauKKYL* 14 27. LauKKYLNSILN* 47 55.
CapNSILN* 68 28. StKYLN* 48 56. LauNSILN* 69
wherein Cap=Caproic acid, Lau=Lauric acid, St=Stearic acid;
Z=Aminocaproic acid, B=Aminolauric acid, X=d, lAla, O=D-Ala; B=dK,
O=dY, Z=dL, X=p=aminoPhe, *=amid, Ol=Oleic acid
B. Synthesis of Cyclic Peptides
I. Cyclic peptides containing intramolecular amide bonds, i.e.
Z=--CONH-- or --NHCO-- of the formulae III and IV as defined above
may be prepared through conventional solid phase synthesis. Thus,
peptide chains may be assembled on the solid support while
incorporating suitable amino and carboxyl side-chain protected
amino acid derivatives at the positions selected for cyclization.
Following completion of peptide chain assembly, the protecting
groups can be selectively removed from the corresponding amino and
carboxyl functions, leaving other protecting groups and the
peptide-support bond intact. Cyclization can then be accomplished
using known peptide coupling agents. Finally, the cyclic peptide
may be cleaved from the support along with deprotection of side
chain moieties using known procedures, and purification of the
desired cyclic peptide can be achieved by chromatographic
techniques.
II. Cyclic peptides containing an intramolecular disulfide bond,
i.e. Z=S--S of the formulae III and IV as defined above may be
prepared through conventional solid phase synthesis while
incorporating suitable S-protected cysteine or homocysteine
residues at the positions selected for cyclization. Following
completion of the chain assembly, two possible routes for
cyclization can be performed: 1. Selective removal of S-protecting
groups with a consequent on-support oxidation of free corresponding
two SH-functions, to form S'--S bonds. This may be followed by
conventional removal of the product from the support and
appropriate chromatographic purification. 2. Removal of the peptide
from the support along with complete side-chain deprotection,
followed by oxidation of free SH-functions in highly dilute aqueous
solution. Both routes lead to the same final desired product.
III. Cyclic peptides containing intramolecular S-alkyl bonds, i.e.
Z=--S(CH.sub.2).sub.t CO--NH or --NH--CO(CH.sub.2).sub.t S-- of the
formulae III and IV as defined above may be prepared through
conventional solid phase synthesis. Thus, an amino acid residue
with a suitable amino-protected side chain, and a suitable
S-protected cysteine or homocysteine residue may be incorporated
during peptide chain assembly at positions selected for
cyclization. The blocked side-chain amino function is selectively
deprotected followed by bromoacylation. The peptide can then be
detached from the support, along with side-chain deprotection,
under acidic conditions. Under neutral or slightly basic
conditions, the corresponding free SH and bromoacylated moieties
may then selectively interact at high dilution to afford the
desired cyclic peptide.
EXAMPLE B1
##STR2##
[General formula (iii) above wherein X.sub.5 =Leu; X.sub.6
=covalent bond; Z=--NH--CO--; m=4, n=1]
Synthesis of the peptide is performed manually on a
p-amethylbenzhydrylamine (MBHA) resin available from Nova,
Switzerland. All solvents, methylene chloride (CH.sub.2 Cl.sub.2),
N-methylpyrrolidone (NMP) and dimethyl sulfoxide (DMSO) are
analytical products of Merck, Germany. Trifluoroacetic acid (TFA),
diisopropylethylamine (DIEA) and N,N'-dicyclohexylcarbodiimide
(DCC) are purchased from Aldrich, U.S.A. 1-Hydroxybenzotriazole
(HOBT) is obtained from Nova, Switzerland. All protected amino acid
derivatives (Boc-AA) are of the L-configuration and are obtained
from Bachem, Switzerland. N.alpha.-amino acid functions are
protected throughout the synthesis by the t-butyloxycarbonyl
(t-Boc) group. Side chain functions are protected as follows: Asp
with 9-fluorenylmethyl (Fm), Lys with 2-chloro-benzyloxycarbonyl,
and at position 1 of the peptide chain with
9-fluorenylmethoxycarbonyl (Fmoc) and Tyr with
2,6-dichlorobenzyl.
The synthesis is initiated by coupling Boc-Asp (OFm) (0.82 g, 2
mmol) to the methylbenzhydryl amine resin (1 g) using DCC (0.42 g,
2 mmol) and HOBT (0.272 g, 2 mmol) as reagents. Loading (0.39
mmol/g) is determined by amino acid analysis. Unreacted residual
amino groups on the polymer are capped by reacting with acetic
anhydride and triethylamine (20 ml and 0.5 ml, correspondingly) in
CH.sub.2 Cl.sub.2 (10 ml). The peptide chain assembly is started
with the Boc-Asp(OFm)-MBHA resin, following the protocol outlined
in Table 1.
TABLE 1 Protocol for manual solid phase synthesis. Step
Reagent/Solvents Time (min.) 1 TFA in CH.sub.2 Cl.sub.2 (30% v/v) 3
2 ThA in CH.sub.2 Cl.sub.2 (50% v/v) 20 3 CH.sub.2 Cl.sub.2 5
.times. 2 4 3% DIEA in CH.sub.2 Cl.sub.2 (v/v) 5 5 3% DIEA in NMP
(v/v) 2 6 NMP 5 .times. 2 7 Ninhydrin test 8 1.6 mmol Boc A.A. +
1.6 ml 1N 45 HOBT + 1.6 ml 1N DCC all in NMP; preactivation - 30
min; filter and add solution to polymer (1 g) 20 DMSO (final vol.
20% v/v) 9 DIEA (6 mmol in NMP) 10 10 NMP 5 11 CH.sub.2 Cl.sub.2 3
.times. 2 12 Ninhydrin test 13 10% Ac.sub.2 O + 3% DIEA in CH.sub.2
Cl.sub.2 5 14 10% Ac.sub.2 O in CH.sub.2 Cl.sub.2 10 15 CH.sub.2
Cl.sub.2 3 .times. 2
Solvents for all washings and reactions are measured to volumes of
10 ml/g resin. All couplings are performed using HOBT active esters
of Boc-amino acid derivatives, prepared by DCC prior to each
coupling step. A molar ratio of 4:1 of Boc-amino acid
1-hydroxybenzotriazole ester (Boc-AA-OBT) and .alpha.-amino group
of a growing peptide chain, respectively, is employed for
couplings. Coupling reactions are monitored by boiling a few mg
(.sup.- 3) of polymer in a solution of ninhydrin in pyridine-water
for 2 min. Coupling of Boc-amino acids is repeated twice to ensure
complete reaction. In the second coupling, half of the amount of
Boc-AA OBT is used. As a rule, after completion of each coupling
step, residual amino groups are capped by treating the resin with
acetic anhydride (10%) and diisopropylethylamine (3%) in methylene
chloride, followed by treatment with 10% acetic acid in methylene
chloride.
Following completion of the peptide chain assembly, the t-Boc
protecting group of Lys-1 is removed, as usual, by 50% TFA in
CH.sub.2 Cl.sub.2 and the newly free .alpha.-amino group is coupled
to stearic acid (0.37 g, 2 mmol) using DCC (0.42 g, 2 mmol) and
HOBT (0.27 g, 2 mmol) as reagents (Protocol). The reaction proceeds
for 120 min and is repeated twice. The OFm and Fmoc side-chain
protecting group of Asp.sup.6 and Lys.sup.1, respectively, are
removed using 50% piperidine in DMF for 1 hr. Following extensive
washings with DMF (3.times.10 ml), dichloromethane (3.times.10 ml),
10% DIEA in CH.sub.2 Cl.sub.2 (3.times.10 ml), DMF (3.times.10 ml)
and CH.sub.2 Cl.sub.2 (3.times.10 ml), the resin is suspended in 7
ml DMF and mixed with five-fold excess (2 mmol) of
(benzotriazolyloxy)tris(dimethyl)aminophosphonium
hexafluorophosphate (BOP) reagent in the presence of seven-fold
excess (2.8 mmol) of DIEA for 8 hr. Cyclization is repeated
following the exact procedure. A negative ninhydrin test indicates
completion of cyclization. The fully assembled cyclic peptide-resin
is washed with CH.sub.2 Cl.sub.2 according to protocol, then dried
under vacuum overnight, over P.sub.2 O.sub.5. Deblocking of
protecting groups and cleavage of the peptide from resin is
achieved by the anhydrous HF technique. Thus, the peptide-resin (1
g) is treated in a Teflon.TM. HF apparatus (Multiple Peptide
System) with 9 ml HF in the presence of a mixture of 1.5 ml of
p-thiocresol and p-cresol (1:1 v/v) for 1 hr at 0.degree. C. The HF
is removed by vacuum and the resin is extracted with peroxide-free
ether (4.times.25 ml), filtered, dried and extracted with 50%
acetic acid in water (3.times.25 ml). Lyophilization of aqueous
filtrate yielded the crude powder of ##STR3##
The crude product was dissolved in 50% aqueous acetic acid and
passed through a Sephadex G-25 column (75.times.2 cm) employing 0.1
N acetic acid as an eluent. Elution was monitored
spectrophotometrically at 274 nm. Lyophilization of the aqueous
solution yielded the peptide free of aromatic additives added as
scavengers at the HF-cleavage step. Yield was 50-70%.
Purification by high performance liquid chromatography (HPLC) is
then carried out on the Sephadex-fractionated products. This can be
performed, however, on the crude peptide. Purifications were
achieved on a Merck RP-8 column (7 .mu.M, 250.times.10 mm). The
peptide is applied in 10% acetonitrile in water and eluted with a
linear gradient established between 0.1% TEA in water and 0.1% TFA
in 75% acetonitrile in water at a flow rate of 10 ml/min. Fractions
are collected and cuts made after inspection by analytical HPLC.
Derived fractions are pooled and lyophilized. Yield of the pure
peptide is 30-35%.
Purity of the product is ascertained by analytical HPLC (Merck
RP-8, 250.times.4 mm column) and amino acid analysis, following
exhaustive acid hydrolysis (6 N HCl), which gives the expected
values of each constituent amino acid.
Other related cyclic derivatives of formulae (iii) and (iv) above
wherein Z is --NH--CO-- or --CO--NH-- are prepared by exactly the
same process while employing the corresponding amino acid
derivatives.
Alternatively, the cyclic derivatives may be prepared by the
processes described above and then the stearoyl or other suitable
lipophilic moiety is introduced into the molecule at the N
terminal.
EXAMPLE B2
##STR4##
[General formula (iii) above wherein X.sub.5 =Leu, X.sub.6 is a
covalent bond, Z=--S--S--; n=1, m=1]
Synthesis of the peptide is performed manually on a
p-methylbenzhydrylamine (MBHA) resin (1 g) as outlined in the
previous example. Cysteine residues, 1 and 6, are introduced into
the peptide chain employing Boc-Cys(S-4-MeBzl)--OH as a building
block. Following completion of the chain assembly and addition of
N-terminal stearoyl moiety, the peptide-resin is treated with
anhydrous HF as described above. The white powder of crude peptide
obtained after lyophilization is dissolved in 0.1% acetic acid
(.sup.- 0.5 mg/ml) and the solution is de-aerated by bubbling
through oxygen-free nitrogen for 2 hr. The pH of the solution is
adjusted with concentrated aqueous NH.sub.4 OH to .about.8.5 and a
solution (.about.1 N) of K.sub.3 Fe(CN).sub.6 (2.5 equiv.) in water
is slowly added dropwise. After complete addition of the oxidant
reagent, the reaction mixture is allowed to stir for .about.1 hr at
room temperature. The solution is then concentrated by rotary
evaporation and the crude cyclic product is fractionated on
Sephadex G-25, as described above. Purification is then achieved by
HPL-C on a Merck RP-8 column (see previous example). Yield of the
pure product is 35-40%. Other cyclic peptides containing S--S
internal bridges are prepared following the above procedure while
employing Boc-Cys(S-4-MeBzl)-OH and Boc-Homocys(S-4-MeBzl)-OH as
building blocks, which are introduced at sites selected for
cyclization.
EXAMPLE B3
##STR5##
[General formula (iii) above wherein X.sub.5 =Leu; X.sub.6
=covalent bond: Z=--S--(CH.sub.2).sub.t --CO--NH--; m=1; n=4;
t=1]
Synthesis of the peptide is performed manually on a
p-methylbenzhydrylamine (MBHA) resin (1 g) as outlined in the
previous example. A cysteine residue 1 is introduced into the
peptide chain employing Boc-Cys(S-4-MeBzl)-OH, while Lys-6 is
introduced as Boc-Lys(.epsilon.-Fmoc)-OH. Following completion of
the chain assembly and addition of N-terminal stearoyl moiety, the
.epsilon.-Fmoc protecting group of Lys-6 is removed by treatment
with 20% piperidine in DMF for 30 min. Extensive washings of the
resin are then performed with DMF (3.times.10 ml), CH.sub.2
Cl.sub.2 (3.times.10 ml), 10% DIEA in CH.sub.2 Cl.sub.2 (3.times.10
ml), DMF (3.times.10 ml) and CH.sub.2 Cl.sub.2 (3.times.10 ml). The
resin is suspended in 10 ml of DMF and mixed with five-fold excess
(2 mmol) of bromoacetic acid anhydride for 6 hr. The resin is
washed with DMF (3.times.10 ml) and the reaction repeated. Negative
ninhydrin test indicates completion of the acylation reaction. The
resin is then washed with DMF (3.times.10 ml), and CHCl.sub.2
(3.times.10 ml), dried in vacuo and treated with anhydrous HF using
anisole (10%) as the only scavenger. A crude bromoacetylated
product is obtained using the same manipulations described in
previous examples. The white powder (.about.0.5 mg/ml) is then
dissolved in 0.1% acetic acid and the pH is adjusted to .about.7.0
by 1 N NaOH. After reacting for 4 hr at room temperature, the
solution, devoid of free SH-functions as indicated by Ellman's
Reagent (Aldrich), is concentrated by rotary evaporation. The crude
product is then purified by being passed through a Sephadex G-25
column, followed by preparative HPLC, as described in Example A.
Yield of the pure product is 25-30%. Other cyclic peptides
containing internal --S--(CH.sub.2).sub.t --CO--NH-- or
--NH--CO--(CH.sub.2).sub.t --S-- bridges are prepared following the
above procedure while employing, at the site selected for
cyclization and elsewhere, the corresponding amino acid
derivatives.
C. The Neurodegenerative Treatment Aspen of the Invention
EXAMPLE C1
Biological Test--Effect of Conjugates of the Invention on the
Survival of .beta.-Amyloid Peptide Treated Neurons
Method
.beta.-Amyloid peptide is known to be involved in Alzheimer's
disease and is a toxic substance to neurons grown in culture (Pike
et al., J. of Nezarosci., 13(4), 1676-1687 (1993); Yankner et al.,
Science, 250:279-282 (1990); Gozes et. al., Proc. Natl. Acad. Sci.
USA, 93:927-432 (1996)).
Rat cerebral cortical cell cultures were prepared by a slight
modification of the techniques described by Forsythe and Westbrook
(J. Physiol. Lord. 365:515, (1988)), in which cerebral cortex was
used instead of hippocampus and newborn rats were utilized instead
of E16 mice. Cerebral cortical cells (1.5-15.times.10.sup.5
cells/35 mm dish) were plated on confluent cerebral cortical
astrocyte feeder cultures as described (Gozes et al., J. Pharmacol
Erp. Therap., 257:959-966 (1991)). The culture medium was
DMEM-(Dulbeco Modified Eagle Medium) containing 5% horse serum and
N3 [media supplement containing a hormone cocktail, according to
(Romijn et al., Brain Res., 254:(4),583-589 (1981))]. After eight
days growth in vitro, the cultures were given a complete change of
medium and then were treated with the .beta.-amyloid peptide (amino
acids 25-35) for 5 days.
The .beta.-amyloid peptide fragment was dissolved in water to a
finalconcentration of 2.5 mM. Experiments were performed with
increasing, doses of the conjugates of the invention (1 mg
initially mixed with 10 .mu.l DMSO and then with a further 10 .mu.l
of DMSO to achieve complete solubilization and diluted in PBS to
obtain a stock solution of 10.sup.-3 M) that were added together
with 25 .mu.M .beta.-amyloid peptide (amino acids 25-35) to
dissociated cerebral cortical cells, nine days after plating of
neurons. Ten .mu.l of conjugate solution was added to 1 ml culture
medium. The duration of treatment was 5 days with no change of
media. After 14 days in culture, cells were fixated for
immunocytochemistry and stained with antibodies against NSE (neuron
specific enolase, a neuronal marker). Neuronal cell counts were
executed on 60 fields. with a total area of 25 mm.sup.2. Neurons
were counted without knowledge of type of treatment as before. Each
value is the mean.+-.SEM of 3 dishes.
The results are shown in FIG. 1 and Table 2. FIG. 1 shows the
summation of five independent experiments, with the control
containing 164, 225, 130, 319, 172 neurons. As can be seen in FIG.
1, St-Lys-Lys-Tyr-Leu-NH.sub.2 is a very active conjugate
exhibiting activities at 10.sup.-13 -10.sup.-9 M with a peak
activity at 10.sup.-12 -10.sup.-10 M. The cell count of cells
treated with both this conjugate and .beta.-amyloid peptide was
higher than the counts of untreated control cells indicating that
this conjugate was able to protect cells also against naturally
occurring death.
TABLE 2 Peptide Activity in the neuronal survival assay, protection
against .beta.-amyloid toxicity: SEQ. ID Active Neuronal % survival
after Concentration Peptide NOS concentration (M) survival (%)
.beta.-amyloid tested 1 St-Lys-Lys-Tyr-Leu-NH.sub.2 6 10.sup.-13
-10.sup.-9 80-110 52 10.sup.-13 -10.sup.-9 2
St-Lys-Lys-Tyr-Val-NH.sub.2 9 10.sup.-13 72 38 10.sup.-13
-10.sup.-9 3 St-Lys-Lys-Tyr-D-Ala-NH.sub.2 10.sup.-15 -10.sup.-12
67-117 44 10.sup.-15 -10.sup.-11 4 St-Lys-Lys-Tyr-Nlc-NH.sub.2 16
10.sup.-14 -10.sup.-12 76-84 44 10.sup.-15 10.sup.-11 5
Lau-Lys-Lys-Tyr-Leu-NH.sub.2 14 10.sup.-11 -10.sup.-10 83-84 63
10.sup.-13 -10.sup.-9 6 Cap-Lys-Lys-Tyr-Leu-NH.sub.2 15 10.sup.-11
73 63 10.sup.-13 -10.sup.-8 7 St-Lys-Tyr-Leu-NH.sub.2 10.sup.-11 96
53 10.sup.-13 -10.sup.-9 8 St-Val-Lys-Lys-Tyr-Leu-NH.sub.2 17
10.sup.-12 -10.sup.-8 70-92 53 10.sup.-13 -10.sup.-8 9
St-Lys-Lys-Tyr-Leu-Nle-NH.sub.2 10 10.sup.-13 -10.sup.-10 95-100 36
10.sup.-13 -10.sup.-9 10 St-Lys-Lys-Tyr-Leu-Asn-NH.sub.2 20
10.sup.-13 -10.sup.-10 52-73 34-57 10.sup.-13 -10.sup.-9 11
St-Lys-Lys-Tyr-Leu-Asn-Ser-NH.sub.2 21 10.sup.-13 83 52 10.sup.-13
-10.sup.-9 12 St-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-NH.sub.2 22 10.sup.-9
123 57 10.sup.-13 -10.sup.-9 13
St-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-NH.sub.2 23 10.sup.-9 97 57
10.sup.-13 -10.sup.-9 14 St-Asn-Ser-Ile-Leu-Asn-NH.sub.2 8
10.sup.-11 10.sup.-10 68-103 40 10.sup.-13 -10.sup.-9 15
St-Ser-Ile-Leu-Asn-NH.sub.2 25 10.sup.-12 -10.sup.-9 84-58 40
10.sup.-13 -10.sup.-9 16 St-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 73
10.sup.-13 -10.sup.-11 100-114 58 10.sup.-13 -10.sup.-9 17
St-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 19 10.sup.-13 -10.sup.-11
88-110 58 10.sup.-13 -10.sup.-9
As can be seen in Table 2, several modifications of the
St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6) conjugate (1) showed
similar activity in protection of neurons from death caused by
.beta.-amyloid as compared to control. In some cases (where
neuronal survival exceed 100% as compared to control) the
conjugates were able to protect also against naturally occurring
death. Notably substitution of Leu of conjugate 1 by Val (3), D-Ala
(3), Nle (4), addition of amino acid residues at the N-terminal (8)
or C-terminal (9,10,11,12,13), deletion of amino acid residues from
the N-terminal (7) or replacement of the lipophilic moiety Stearoyl
by Lauroyl (Lau), (5) or Caproyl (Cap,6) resulted in conjugates
with activity similar to St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID
NO:6).
EXAMPLE C2
Effects of a Conjugate of the Invention on Learning and Memory in
Animal Models of Alzheimer (Morris Water Maze)
In vivo Model for Cholinergic Inhibition. 18 Male rats (Wistar,
250-300 g) were injected intracerebroventricularly (i.c.v.) at a
rate of 0.21 .mu.l/min., using plastic tubing (PE-20) attached to
25G needle; controls received an injection of saline 2 .mu.l/side,
experimental animals received injections of the cholinergic blocker
(ethylcholine aziridium) AF64A (3 nmol/2 .mu.l/side).
Drug treatment was initiated 7-10 days after AF64A injection.
Animals were divided into two equal groups. Test group received
daily nasal administration of St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID
NO:6) dissolved in 10% Sefsol.TM. and 40% isopropanol at a
concentration of 10 .mu.g/40 .mu.l (20 .mu.l administered through
each nostril). Control animals received intranasal administration
of the vehicle. The rats were partially anesthetized by
diethylether prior to nasal administration. Following seven days of
drug administration, behavioral assays were conducted for an
additional 10 days. Drugs were applied by nasal administration 1
hour prior to testing. All animals were chronically treated (every
two days) with 50,000 units of durabiotic antibiotics to avoid
infection.
Learning test procedure was carried out according to the Morris
Water Maze procedure (Morris et al., Nature, 297:681-683, 1982;
Morris et al., Nature, 319:774-776, 1986).
1. Administration of St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6)
Rats were placed in a circular pool, 1.26 m in diameter, 0.2 m
deep, equipped with a clear plexiglas column, with a 13.3 cm
platform reaching just below the surface of the water
(22-24.degree. C). Drugs were applied daily by nasal administration
1 hour prior to testing. The latency of reaching the platform was
recorded for each rat (in seconds) and the changes over days of
training were graphed, which reflect learning and memory.
As can be seen in FIG. 2, control animals injected with AF64A
(.largecircle.) showed a smaller improvement in the latency of
reaching the platforms compared with animals injected with AF64A
and nasally administered with St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID
NO:6) (.circle-solid.). These results indicate that the conjugate
of the invention is able to improve learning and memory in an
animal model of Alzheimer.
II. Administration of St-Lys-Lys-Tyr-D-Ala-NH.sub.2
FIG. 3 depicts the effects of St-Lys-Lys-Tyr-D-Ala-NH.sub.2
(indicated KKYO in the figure) on learning and memory in the
Alzheimer's in vivo test (AF64A-cholinotoxicity test) as described
above. The results obtained were similar to those described above
with control animals which were treated only with AF64A
(.circle-solid.) showed smaller improvement in the latency of
reaching the platforms compared with animals nasally administered
also with St-Lys-Lys-Tyr-D-Ala-NH.sub.2 (.tangle-solidup.). An
additional probe test was also performed (Gozes et al., Proc. Nat.
Acad. Sci. USA 93:427-432, 1996), in this test: after the animals
know where the platform is in the water pool, the platform is
removed and the time the animals spend in the area occupied before
by the platform is recorded, this time periods represents memory
retention of a previously studied test. As observed in FIG. 4,
St-Lys-Lys-Tyr-D-Ala-NH.sub.2 treated animals exhibit better memory
retention than the AF64A treated animals that did not receive the
peptide. Following this test the platform is placed back in the
water pool, but this time it is visible to the swimming rat and the
time required to reach it is measured. In this case the parameter
measured is possible motor deficits. As can be seen in FIG. 5,
there are no differences between the groups, thus overall, the test
measured learning and memory and not motor changes.
EXAMPLE C3
Effects of a Conjugate of the Invention on Learning and Memory of
Normal Animals
The experiment described in Example C2 was repeated but the animals
were injected with saline instead of with AF64A and thus were
normal animals, not featuring Alzheimer-like cognitive damages. The
animals were divided into two groups, one administered intranasally
only with the vehicle (Sefsol+isopropanol as described above) and
one receiving intranasal administration of
St-Lys-Lys-Tyr-D-Ala-NH.sub.2. As can be seen in FIG. 6, an
apparent somewhat faster learning was observed in the peptide
treated group indicating a possible improvement of cognitive
function also in undamaged, normal animals.
EXAMPLE C4
Effects of a Conjugate of the Invention on a Model for Memory
Retention
A new model was developed in order to assess the capability of
animals to retain the memory of a previously learned test. Animals
(N=5-10) were first taught to find a submerged platform in a water
maze as described above. For assessment of memory retention, the
animals were subjected to daily swim (a test a day) in order to
learn to find the hidden platform. After a week, the animals which
showed the highest score in the study test, were chosen for the
experiment. AF64A or saline were injected into the third ventricle
of the brain and following a week of recuperation, the animals were
treated by intranasal administration of the peptide
St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6) as described above.
Following a week of peptide treatment, the animals were tested
again in the water maze and the experiment of retention of the
learned task (i.e. finding the platform) was repeated as follows:
the animal was placed on a platform for 1 min. And then placed in
the water for swimming to the platform and the time required to
reach the platform was measured. The results of the first swim are
summarized in FIG. 7. As can be seen in the figure, it is evident
that animals treated with the peptide are protected from memory
loss, and are capable of retaining the memory of the task studied
previous to the injection with AF64A since they behave in a similar
manner to control.
After 1 min. on the platform, the animal is placed back in the
water for an additional swim and search of the hidden platform.
FIG. 8 shows the result for the second swim. As can be seen, FIG. 8
also shows an improvement in learning and memory in animals treated
with the peptide. This model, is the first demonstration of the
activity of the peptide and facilitating memory retention.
Finally, the platform is removed and the time spent by the animals
at the area where the platform is featured in FIG. 9. The results
summarized above, clearly demonstrate that
St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6) is involved in learning,
in working memory, and in memory retention.
EXAMPLE C5
Effect of Conjugates of the Invention on a Model for Mental
Retardation
A Model for Mental Retardation: Apolipoprotein E-deficient Mice
It has recently been discovered that mice deficient in
apolipoprotein E (ApoE) (Plump et al., Cell, 71:343-363 (1992)) are
retarded in their acquisition of developmental milestones. The ApoE
deficient mice were tested for the development of behavioral
milestones and were found to be significantly retarded in their
acquisition of forelimb placing behavior (postnatal day 11-13) as
compared to control animals (postnatal day 2-5). A two day delay in
the acquisition of cliff aversion behavior also was observed in
these mice.
8 newborn normal mice were injected (s.c. 1.2 .mu.g 120 .mu.u) with
St-Lys-Lys-Tyr-Leu-NH.sub.2 or with saline. 8 ApoE deficient
newborn mice were treated similarly.
The results of forelimb placing behavior acquisition are shown in
FIGS. 10 and 11. As can be seen ApoE deficient mice treated with
St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6) or with the known
St-Nle.sup.17 -VIP-NH.sub.2 (shown for comparison only)
St-Nle.sup.17 -VIP improved their placing acquisition essentially
to the level of control as compared with untreated ApoE deficient
mice.
FIGS. 12 and 13 show cliff avoidance acquisition in animals treated
as above. As can be seen, ApoE deficient mice treated with
St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6) or St-Nle-V1P -NH.sub.2
(shown for comparison only) showed even better cliff avoidance
acquisition than normal controls or similar cliff avoidance
behavior to control.
EXAMPLE C6
Effect of a Conjugate of the Invention on the Cholinergic Activity
in Apolipoprotein E Deficient Mice
Apo-E deficient mice were assayed for their cholinergic activity as
described by Fonnum, F. A., J Neurochem., 24:413-415, 1975.
Briefly, brains from 21 days normal (control) and ApoE-deficient
mice were assessed by measuring their Chat activity. Choline acetyl
transferase (Chat) activity was determined as described before (see
text), by measurements of the rate of synthesis of
(14Cqacctylcholine from choline and [14C]acetylCoA. Non-specific
background was measured in the absence of choline. Each brain
(300-400 mg) was homogenized in a teflon homogenizer, with 10
volumes of 50 mM phosphate buffer (pH=7.4) containing 300 mM NaCl,
30 mM EDTA and 0.5% triton. The homogenates were centrifuged at
12000 g for 15 min., and 10 141 of the supernatant (in triplicates)
were mixed with 10 .mu.l of a solution containing: 14 .mu.M
14C-acetyl-CoA (56 mCi/mmol NEN), 20 mM Acetylcholine, 1.6 mM
choline chloride, 0.25 mM eserine, and phosphate buffer. The
reaction was carried out at 37.degree. C. for 15 min. stopped by
adding 50 .mu.l of 15 mg/ml tetraphenylboron prepared in
3-heptanone and mixed in a vortex for 30 sec. Twenty .mu.l of the
organic phase were collected after 2 min. microfuging, then were
mixed with scintillation liquid and radioactivity was measured in a
beta-counter.
Experiments were conducted on 21-day-old animals chronically
injected with peptides or with saline. Injection is performed
subcutaneously, peptides are dissolved (100 .mu.g/30 .mu.l) in DMSO
and diluted with saline to obtain the desired concentrations. Day
1-4: 4 .mu.g peptide/20 .mu.l saline; day 5-10: 8 .mu.g/40 .mu.l
saline; day 11-14: 16 .mu.g/80 .mu.l saline.
The results of cholinergic activity show a reduction in Apo-E
deficient mice. The cholinergic activity of mice administered with
St-Lys-Lys-Tyr-Leu (SEQ ID NO:6), are shown in FIG. 14. The figure
demonstrates ApoE mice treated with St-Lys-Lys-Tyr-Leu-NH.sub.2
(SEQ ID NO:6) which show an increase in choline acetyl transferase
activity as compared to the control levels.
FIG. 14 indicate that St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6)
increases cholinergic activity in the apolipoprotcin E deficient
mice (100% activity indicates 669-758 pmole/mg, protein/min. in all
the determinations of choline acetyl transferase activity).
EXAMPLE C7
Biodistribution of St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6)
St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6) was radioiodinated by
using the chloramine T methods as described in Gozes et al.,
Endocrinology, 134:2121-2125 (1994) and about 7.8.times.10.sup.6
cpm/2 .mu.l 5% Sefsol.TM., 20% isopropanol/rat were applied
intranasally to 250-300 g rats. Animals were sacrificed 30 minutes
following drug administration and the frontal cortex was removed,
weighed and counted for radioactivity in a gamma counter.
Radioactive tissue samples (containing 1400 cpm/gram sample) were
thereafter homogenized and subjected to centrifugation (5,400 g for
25 min.). Supernatants were then subjected to HPLC analysis against
St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6) as a marker (eluting
using an acetonitrile gradient at fraction 25). Samples were
monitored for radioactivity in a gamma counter.
As can be seen in FIG. 15, intact St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ
ID NO:6) could be observed in the brain thirty minutes after
application, indicating that the conjugates of the invention, when
administered intranasally are able to reach the brain.
D. The Impotence Prevention Aspect of the Invention
Biological Tests for Penile Reflexes
The biological tests involved measurements of penile reflexes in
castrated rats following transdermal application of the conjugates
of the invention. In a first type of biological experiment the
effects of compositions with various carriers on penile reflexes
were measured and it was found that Sefsol.TM. was the most
effective carrier.
(a) Methods
Animal Model for Impotence
Rats with reduced sexual potential due to castration were employed.
Male rats (250-300 g, about three months old) were kept in a
12-hours light, 12-hours dark cycle. Experiments were always
conducted within the dark period, 2-6 hours after the onset of
darkness. Male rats were castrated and given partial testosterone
replacement (4 .mu.g/100 g body weight) in the form of daily
injection during 14-21 consecutive days (the duration of the
experiment). Experiments were conducted one week following
surgery.
Direct Evaluation of Penile Reflexes (Erections)
A procedure was utilized that employed the technique that measures
sexual reflexes in the penis, which enables direct evaluation of
penile erection following transdermal administration of the drug.
Successful reproduction depends, in large part, upon the precise
execution of temporally organized, functionally related behavioral
units. In these experiments, we concentrated on the final stages of
the erection process (reddening of the penis accompanied by its
distension and extension leading to complete erection) and
monitored the latency time to the first E2 and first cup (Okumura,
M., et al., Chem. Pharm. Bull. 37, 1375 (1989)).
For testing, each animal was restrained in a supine position with
the anterior portion of its body enclosed in a loosely fitting
cylinder (7 cm diam.). After a belt was secured around the torso,
the glans penis was extruded from its sheath and gently held
perpendicular to the abdomen by a thin wooden applicator positioned
at the posterior of the penis. The legs of the male were held by
the observer and this position was maintained throughout the test
period. The duration of the session was 45 minutes. The latencies
and numbers of E2 and cups were recorded and plotted.
An E2 is defined as a complete erection which can be followed by
cup in which the penile tip is turned into a cup-like structure,
whereby the glands flare out such that the penis is wider in its
distal portion than its base. This final stage requires E2 and is
probably a pre-requisite for ejaculation. Using all the parameters
one can obtain a reliable measure of the sexual activity of the
tested rat.
Duration of ES2 was determined by monitoring animals for a period
of 45 minutes and calculating total time of erection whether by
measuring the length of a single erection episode or by adding
together several non-continuous erection episodes. The minimal
duration of a single erection episode was calculated as half a
minute.
EXAMPLE D1
Effect of the Conjugates of the Invention in a Rat Model of
Impotence
Conjugates were dissolved in dimethylsulfoxide (DMSO) at a
concentration of 10.sup.-3 M and 10 .mu.l were utilized per
application. Rats used were castrated-treated by partial
testosterone replacement (Gozes et al., J. Clin. Inves., 90:810-814
(1992)). In short, male rats (90-100 days old) were castrated and
immediately injected daily with 4 .mu.g/100 g BW testosterone, s.c.
for 21 consecutive days, the duration of the experiment.
Experiments were initiated one week after surgery. Penile reflexes
were measured as described above.
FIG. 16 shows that peptide 6
(St-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID
NO:24) was able to significantly improve both number of cups and
the number of E2 as compared to control. FIG. 17 depicts peptide 6
in comparison to peptide 26. Experiments were repeated for the
other conjugates of the invention substantially as described above,
but peptides were dissolved in 5% Sefsol.TM. and 20% isopropanol to
a final concentration of 7 .mu.g/10 .mu.l per animal. The
conjugates tested were: St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6);
St-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID NO:25);
St-Asn-Ser-Ile-Tyr-Leu-Asn-NH.sub.2 (SEQ ID NO:74);
St-Lys-Lys-Tyr-D-Ala-NH.sub.2 ; St-Lys-Lys-Tyr-Val-NH.sub.2 (SEQ ID
NO:9); St-Lys-Lys-Tyr-Leu-Nle-NH.sub.2 (SEQ ID NO:10);
St-Lys-Lys-Tyr-Leu-Pro-Pro-Asn-Ser-Ile-Leu-Asn-NH.sub.2 (SEQ ID
NO:73).
The results are shown in FIGS. 18-27 and indicate that all tested
conjugates were able to improve all tested impotence parameters
which were determined (No. of cups, No. of E2 and reduction of
latency to the first cup) as compared to control.
EXAMPLE D2
Biodistribution Following Topical Administration of
St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6)
St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6) was radioiodinated as
before and about 2.2.times.10.sup.6 cpm/2 .mu.l 5% Sefsol.TM., 20%
isopropanol/rat were applied topically on the sex organ of 250-300
g rats. Animals were sacrificed at indicated times and tissues were
weighed and counted for radioactivity in a gamma counter.
The results are shown in FIG. 28. These results indicate that
conjugates which were administered transdermally were able to
penetrate the inner tissue of the animal.
EXAMPLE D3
HPLC Analysis of Intestinal Extract
Experiment was performed as indicated in Example D2. Animals were
sacrificed 30 minutes following drug administration and the
intestine was removed weighed and counted for radioactivity in a
gamma counter. Radioactive tissue samples were thereafter
homogenized and subjected to centrifugation (5,400 g for 25 min.).
Supernatants were then subjected to HPLC analysis against
radiolodinated St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6) as a
marker (eluting using an acetonitrile gradient at fraction 25).
Samples were monitored for radioactivity in a gamma counter.
The results are shown in FIG. 29. As can be seen from the results
in FIGS. 26 and 27 of the applied 2.2.times.10.sup.6 cpm/g more
than 80,000 cpm/g were located in the intestine 30 mins. following
application. By comparison, using the same amounts of radiolabeled
St-Nle.sup.17 -VIP only 3700 cpm/g were obtained in the intestine
(Gozes et al., Endocrinology, 134:2121-2125 (1994)) showing that
the short conjugate of the invention has a much better penetration
than the full 28 amino acid conjugate.
EXAMPLE D4
Effect of the Conjugates of the Invention in a Rabbit Model of
Impotence
New Zealand white rabbits (from Yokneam, Israel) were anesthetized
with Rompun and Ketavet. Additionally, pentobarbital 10 mg/kg, i.v.
was administered into an ear vein. Anesthesia was maintained by
bolus injections of pentobarbital (5-10 mg/kg). The animals were
placed in a supine position on an operating table, in a
temperature-regulated environment. The area around the penis was
shaved and a 20 gauge needle was inserted into the left or right
corpus cavernosum and the catheter connected to a pressure
transducer for a continuous recording of the intracavarnosal (i.c.)
pressure. The transducer amplifier used was Model PM-1000 (CWE
incorporated); System 1000 power supply (CWE incorporated);
Software: DI 200 PGH/PGL (DATA Q Instrument Inc.). When injection
was required, a second catheter was placed into the other side of
the corpus cavernosum for the administration of the drugs, both
catheters were filled with blood. The catheter for blood pressure
recording was flushed with 0.5 ml 2% heparin. Increases in the i.c.
blood pressure are expressed on the graphs as mm Hg.
FIG. 30 shows preliminary results of change of pressure obtained
with injections of St-Lys-Lys-Tyr-Leu-NH.sub.2 (SEQ ID NO:6), in
the corpus cavernosum. Here 10 .mu.g showed increased activity,
with penile blood pressure increasing to 75 mmHg (from 15-20 mmHg)
indicating that this conjugate can effect penile erection also by
direct injection.
E. Pharmaceutical Compositions for Transdermal Applications
An example of ointment composition for transdermal application of
lipophilic conjugated peptides in accordance with the present
invention with Sefsol 318.TM. as a carrier comprises: 1 mg peptide
per 714 .mu.l 10% Sefsol 318.TM. (glyceryl monocaprylate) and 714 ,
.mu.l 40% isopropanol (final concentrations: 5% Sefsol, 20%
isopropanol and about 0.7 mg/ml peptide).
SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 80 <210>
SEQ ID NO: 1 <211> LENGTH: 28 <212> TYPE: PRT
<213> ORGANISM: HOMO SAPIENS <400> SEQUENCE: 1 His Ser
Asp Ala Val Phe Tyr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15
Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn 20 25 <210>
SEQ ID NO: 2 <211> LENGTH: 25 <212> TYPE: PRT
<213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: Any one or all
of residues 1, 2 and 3 may be present or absent; residue 1 is Ala,
Val, or absent; residue 2 is Ala, Val, or absent; residue 3 is Ala,
Val, Lys, D-Lys, Orn or absent; one of residues 1, 2, 3 or 4 is the
N-terminal residue, and may be modified with a lipophilic moiety;
residue 4 is Lys, D-Lys, or Orn; residue 5 is Tyr, D-Tyr, Phe, Trp,
or p-amino Phe; residue 6 is a hydrophobic amino acid residue; any
one or all of residues 7-11 may be present or absent; residue 7 is
Asn, Ser, Ile, Tyr, Leu, Nle, D-Ala, or absent; residue 8 is Ser or
absent; residue 9 is Ile, Tyr, or absent; residue 10 is Leu or
absent; residue 11 is Asn or absent; any one or all of residues
12-25 may be present or absent; residue 12 is a Ser, Asn, a
hdrophobic aliphatic amino acid residue, Tyr, Lys, Val, Ala, a
non-charged amino acid residue, or absent; residue 13 is Ile, Tyr,
Asn, a hydrophobic aliphatic amino acid residue, Lys, D-Lys, Orn,
Ala, Val, a non- charged amino acid residue, or absent; residue 14
is Leu, Asn, a hydrophobic aliphatic amino acid residue, Tyr, Lys,
a non-charged amino acid residue, Ala, Val, D-Lys, Orn, D-Tyr, Phe,
Trp, p-amino Phe, or absent; residue 15 is Asn, a hydrophobic
aliphatic amino acid residue, Tyr, Lys, D-Lys, Orn, D-Tyr, Phe,
Trp, p-amino Phe, Ala, Val, or absent; residue 16 is Asn, a
hydrophobic aliphatic amino acid residue, Tyr, Lys, Val, Ala,
D-Tyr, Phe, Trp, p-amino Phe, Nle, Leu, Ile, Ser, D-Ala, D-Lys,
Orn, or absent; residue 17 is a hydrophobic aliphatic amino acid
residue, Lys, D-Lys, Orn, Asn, Ser, Ile, Leu, Tyr, Nle, D-Ala,
D-Tyr, Phe, Trp, p-amino Phe, or absent; residue 18 is Asn, Lys,
D-Lys, Orn, Tyr, D-Tyr, Phe, Trp, p-amino Phe, Ser, Ile, Leu, Nle,
D-Ala, a hydrophobic aliphatic amino acid residue, or absent;
residue 19 is Ser, Tyr, D-Tyr, Phe, Trp, p-amino Phe, a hydrophobic
aliphatic amino acid residue, Ile, Leu, Asn, Nle, D-Ala, or absent;
residue 20 is Ile, Tyr, Ser, Asn, Leu, a hydrophobic aliphatic
amino acid residue, Nle, D-Ala, or absent; residue 21 is Leu, Ile,
Tyr, Asn, Ser, Nle, D-Ala, or absent; residue 22 is Asn, Ile, Tyr,
Ser, Leu, or absent; residue 23 is Asn, Leu, Ile, Tyr, or absent;
residue 24 is Asn, Leu, or absent; residue 25 is Asn or absent; the
C-terminal residue is amidated or modified with a lipophilic
moiety. <400> SEQUENCE: 2 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 <210> SEQ ID NO: 3 <211> LENGTH: 25
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: Any one or all of residues 1-7 may be present or
absent; residue 1 is Val, Ala, or absent; residue 2 is Val, Ala, or
absent; residue 3 is Lys, Val, or absent; residue 4 is Lys or
absent; residue 5 is Tyr, Lys, or absent; residue 6 is a
hydrophobic aliphatic amino acid residue, Tyr, Lys, or absent;
residue 7 is Asn, a hydrophobic aliphatic amino acid residue, or
absent; residue 9 is Ile or Tyr; any one or all of residues 12-25
may be present or absent; residue 12 is a Ser, Asn, a hdrophobic
aliphatic amino acid residue, Tyr, Lys, Val, Ala, a non-charged
amino acid residue, or absent; residue 13 is Ile, Tyr, Asn, a
hydrophobic aliphatic amino acid residue, Lys, D-Lys, Orn, Ala,
Val, a non- charged amino acid residue, or absent; residue 14 is
Leu, Asn, a hydrophobic aliphatic amino acid residue, Tyr, Lys, a
non-charged amino acid residue, Ala, Val, D-Lys, Orn, D-Tyr, Phe,
Trp, p-amino Phe, or absent; residue 15 is Asn, a hydrophobic
aliphatic amino acid residue, Tyr, Lys, D-Lys, Orn, D-Tyr, Phe,
Trp, p-amino Phe, Ala, Val, or absent; residue 16 is Asn, a
hydrophobic aliphatic amino acid residue, Tyr, Lys, Val, Ala,
D-Tyr, Phe, Trp, p-amino Phe, Nle, Leu, Ile, Ser, D-Ala, D-Lys,
Orn, or absent; residue 17 is a hydrophobic aliphatic amino acid
residue, Lys, D-Lys, Orn, Asn, Ser, Ile, Leu, Tyr, Nle, D-Ala,
D-Tyr, Phe, Trp, p-amino Phe, or absent; residue 18 is Asn, Lys,
D-Lys, Orn, Tyr, D-Tyr, Phe, Trp, p-amino Phe, Ser, Ile, Leu, Nle,
D-Ala, a hydrophobic aliphatic amino acid residue, or absent;
residue 19 is Ser, Tyr, D-Tyr, Phe, Trp, p-amino Phe, a hydrophobic
aliphatic amino acid residue, Ile, Leu, Asn, Nle, D-Ala, or absent;
residue 20 is Ile, Tyr, Ser, Asn, Leu, a hydrophobic aliphatic
amino acid residue, Nle, D-Ala, or absent; residue 21 is Leu, Ile,
Tyr, Asn, Ser, Nle, D-Ala, or absent; residue 22 is Asn, Ile, Tyr,
Ser, Leu, or absent; residue 23 is Asn, Leu, Ile, Tyr, or absent;
residue 24 is Asn, Leu, or absent; residue 25 is Asn or absent; the
C-terminal residue is amidated or modified with a lipophilic
moiety. <400> SEQUENCE: 3 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa
Leu Asn Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 <210> SEQ ID NO: 4 <211> LENGTH: 25
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: Residue 1 is Asp, Glu, Cys, Orn, Lys, or an amino acid
with a side chain of -(CH2)2NH3, and residue 1 may be modified with
a lipophilic moiety; residues 1 and 6 are linked by a -CO-NH-,
-NH-CO-, -S-S-, -S(CH2)tCO-NH-, or -NH-CO(CH2)tS- bond; residue 5
is a hydrophobic aliphatic amino acid residue; residue 6 is Asp,
Glu, Cys, Orn, Lys, or an amino acid with a side chain of
-(CH2)2NH3; any one or all of residues 7-11 may be present or
absent; residue 7 is Asn, Ser, Ile, Tyr, Leu, or absent; residue 8
is Ser or absent; residue 9 is Ile, Tyr, or absent; residue 10 is
Leu or absent; residue 11 is Asn or absent; any one or all of
residues 12-25 may be present or absent; residue 12 is a Ser, Asn,
a hdrophobic aliphatic amino acid residue, Tyr, Lys, Val, Ala, a
non-charged amino acid residue, or absent; residue 13 is Ile, Tyr,
Asn, a hydrophobic aliphatic amino acid residue, Lys, D-Lys, Orn,
Ala, Val, a non- charged amino acid residue, or absent; residue 14
is Leu, Asn, a hydrophobic aliphatic amino acid residue, Tyr, Lys,
a non-charged amino acid residue, Ala, Val, D-Lys, Orn, D-Tyr, Phe,
Trp, p-amino Phe, or absent; residue 15 is Asn, a hydrophobic
aliphatic amino acid residue, Tyr, Lys, D-Lys, Orn, D-Tyr, Phe,
Trp, p-amino Phe, Ala, Val, or absent; residue 16 is Asn, a
hydrophobic aliphatic amino acid residue, Tyr, Lys, Val, Ala,
D-Tyr, Phe, Trp, p-amino Phe, Nle, Leu, Ile, Ser, D-Ala, D-Lys,
Orn, or absent; residue 17 is a hydrophobic aliphatic amino acid
residue, Lys, D-Lys, Orn, Asn, Ser, Ile, Leu, Tyr, Nle, D-Ala,
D-Tyr, Phe, Trp, p-amino Phe, or absent; residue 18 is Asn, Lys,
D-Lys, Orn, Tyr, D-Tyr, Phe, Trp, p-amino Phe, Ser, Ile, Leu, Nle,
D-Ala, a hydrophobic aliphatic amino acid residue, or absent;
residue 19 is Ser, Tyr, D-Tyr, Phe, Trp, p-amino Phe, a hydrophobic
aliphatic amino acid residue, Ile, Leu, Asn, Nle, D-Ala, or absent;
residue 20 is Ile, Tyr, Ser, Asn, Leu, a hydrophobic aliphatic
amino acid residue, Nle, D-Ala, or absent; residue 21 is Leu, Ile,
Tyr, Asn, Ser, Nle, D-Ala, or absent; residue 22 is Asn, Ile, Tyr,
Ser, Leu, or absent; residue 23 is Asn, Leu, Ile, Tyr, or absent;
residue 24 is Asn, Leu, or absent; residue 25 is Asn or absent; the
C-terminal residue is amidated or modified with a lipophilic
moiety. <400> SEQUENCE: 4 Xaa Lys Lys Tyr Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 20 25 <210> SEQ ID NO: 5 <211> LENGTH: 25
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: Any one or all of residues 1-6 may be present or
absent; one of residues 1-6 is the N-terminal residue and may be
modified with a lipophilic moiety; residue 1 is Ala or absent;
residue 2 is Val, Ala, or absent; residue 3 is Lys or absent;
residue 4 is Lys or absent; residue 5 is Tyr, Lys, or absent;
residue 6 is a hydrophobic aliphatic amino acid residue, Tyr, Lys,
or absent; residue 7 is Asp, Glu, Cys, Orn, Lys, or an amino acid
with a side chain of -(CH2)2NH3; residues 7 and 13 are linked by a
-CO-NH-, -NH-CO-, -S-S-, -S(CH2)tCO-NH-, or -NH-CO(CH2)tS- bond;
residue 8 is Asn or absent; residue 10 is Ile or Tyr; residue 13 is
Asp, Glu, Cys, Orn, Lys, or an amino acid with a side chain of
-(CH2)2NH3; any one or all of residues 14-27 may be present or
absent; residue 14 is a Ser, Asn, a hdrophobic aliphatic amino acid
residue, Tyr, Lys, Val, Ala, a non-charged amino acid residue, or
absent; residue 15 is Ile, Tyr, Asn, a hydrophobic aliphatic amino
acid residue, Lys, D-Lys, Orn, Ala, Val, a non- charged amino acid
residue, or absent; residue 16 is Leu, Asn, a hydrophobic aliphatic
amino acid residue, Tyr, Lys, a non-charged amino acid residue,
Ala, Val, D-Lys, Orn, D-Tyr, Phe, Trp, p-amino Phe, or absent;
residue 17 is Asn, a hydrophobic aliphatic amino acid residue, Tyr,
Lys, D-Lys, Orn, D-Tyr, Phe, Trp, p-amino Phe, Ala, Val, or absent;
residue 18 is Asn, a hydrophobic aliphatic amino acid residue, Tyr,
Lys, Val, Ala, D-Tyr, Phe, Trp, p-amino Phe, Nle, Leu, Ile, Ser,
D-Ala, D-Lys, Orn, or absent; residue 19 is a hydrophobic aliphatic
amino acid residue, Lys, D-Lys, Orn, Asn, Ser, Ile, Leu, Tyr, Nle,
D-Ala, D-Tyr, Phe, Trp, p-amino Phe, or absent; residue 20 is Asn,
Lys, D-Lys, Orn, Tyr, D-Tyr, Phe, Trp, p-amino Phe, Ser, Ile, Leu,
Nle, D-Ala, a hydrophobic aliphatic amino acid residue, or absent;
residue 21 is Ser, Tyr, D-Tyr, Phe, Trp, p-amino Phe, a hydrophobic
aliphatic amino acid residue, Ile, Leu, Asn,
Nle, D-Ala, or absent; residue 22 is Ile, Tyr, Ser, Asn, Leu, a
hydrophobic aliphatic amino acid residue, Nle, D-Ala, or absent;
residue 23 is Leu, Ile, Tyr, Asn, Ser, Nle, D-Ala, or absent;
residue 24 is Asn, Ile, Tyr, Ser, Leu, or absent; residue 25 is
Asn, Leu, Ile, Tyr, or absent; residue 26 is Asn, Leu, or absent;
residue 27 is Asn or absent; the C-terminal residue is amidated or
modified with a lipophilic moiety. <400> SEQUENCE: 5 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Leu Asn Xaa Xaa Xaa Xaa 1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 <210> SEQ ID NO: 6
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism:Artificial <220> FEATURE:
<223> OTHER INFORMATION: The N-terminus is modified with a
stearoyl moiety; the C-terminal residue is amidated. <400>
SEQUENCE: 6 Lys Lys Tyr Leu 1 <210> SEQ ID NO: 7 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Unknown
<220> FEATURE: <223> OTHER INFORMATION: Description of
Unknown Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated. <400> SEQUENCE: 7 Ala Val Lys
Lys Tyr Leu 1 5 <210> SEQ ID NO: 8 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated. <400> SEQUENCE: 8 Asn Ser Ile
Leu Asn 1 5 <210> SEQ ID NO: 9 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated. <400> SEQUENCE: 9 Lys Lys Tyr
Val 1 <210> SEQ ID NO: 10 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:
<223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated; residue 5 is Nle. <400>
SEQUENCE: 10 Lys Lys Tyr Leu Xaa 1 5 <210> SEQ ID NO: 11
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism:Artificial <220> FEATURE:
<223> OTHER INFORMATION: The N-terminus is modified with a
stearoyl moiety; the C-terminal residue is amidated. <400>
SEQUENCE: 11 Asn Ser Tyr Leu Asn 1 5 <210> SEQ ID NO: 12
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism:Artificial <220> FEATURE:
<223> OTHER INFORMATION: The N-terminus is modified with a
stearoyl moiety; the C-terminal residue is amidated. <400>
SEQUENCE: 12 Asn Ser Ile Tyr Asn 1 5 <210> SEQ ID NO: 13
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism:Artificial <220> FEATURE:
<223> OTHER INFORMATION: The N-terminus is modified with a
stearoyl moiety; the C-terminal residue is amidated. <400>
SEQUENCE: 13 Lys Lys Tyr Leu Pro Pro Asn Ser Ile Leu Asn 1 5 10
<210> SEQ ID NO: 14 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The N-terminus
is modified with a lauroyl moiety; the C-terminal residue is
amidated. <400> SEQUENCE: 14 Lys Lys Tyr Leu 1 <210>
SEQ ID NO: 15 <211> LENGTH: 4 <212> TYPE: PRT
<213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The N-terminus
is modified with a caproyl moiety; the C-terminal residue is
amidated. <400> SEQUENCE: 15 Lys Lys Tyr Leu 1 <210>
SEQ ID NO: 16 <211> LENGTH: 4 <212> TYPE: PRT
<213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The N-terminus
is modified with a stearoyl moiety; the C-terminal residue is
amidated; residue 4 is Nle. <400> SEQUENCE: 16 Lys Lys Tyr
Xaa 1 <210> SEQ ID NO: 17 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:
<223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated. <400> SEQUENCE: 17 Val Lys
Lys Tyr Leu 1 5 <210> SEQ ID NO: 18 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated. <400> SEQUENCE: 18 Leu Asn
Ser Ile Leu Asn 1 5 <210> SEQ ID NO: 19 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated. <400> SEQUENCE: 19 Tyr Leu
Asn Ser Ile Leu Asn 1 5 <210> SEQ ID NO: 20 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Unknown
<220> FEATURE: <223> OTHER INFORMATION: Description of
Unknown Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated. <400> SEQUENCE: 20 Lys Lys
Tyr Leu Asn 1 5 <210> SEQ ID NO: 21 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated. <400> SEQUENCE: 21 Lys Lys
Tyr Leu Asn Ser 1 5 <210> SEQ ID NO: 22 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated. <400> SEQUENCE: 22 Lys Lys
Tyr Leu Asn Ser Ile 1 5 <210> SEQ ID NO: 23 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Unknown
<220> FEATURE: <223> OTHER INFORMATION: Description of
Unknown Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated. <400> SEQUENCE: 23 Lys Lys
Tyr Leu Asn Ser Ile Leu 1 5 <210> SEQ ID NO: 24 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Unknown
<220> FEATURE: <223> OTHER INFORMATION: Description of
Unknown Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated; <400> SEQUENCE: 24 Ala Val
Lys Lys Tyr Leu Asn Ser Ile Leu Asn 1 5 10 <210> SEQ ID NO:
25 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Unknown <220> FEATURE:
<223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated. <400> SEQUENCE: 25 Ser Ile
Leu Asn 1 <210> SEQ ID NO: 26 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a caproyl moiety; the
C-terminal residue is amidated; residues 5 and 6 are aminocaproic
acid or D-Leu. <400> SEQUENCE: 26 Lys Lys Tyr Leu Xaa Xaa 1 5
<210> SEQ ID NO: 27 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The N-terminus
is modified with a lauroyl moiety;the C-terminal residue is
amidated; residue 5 is aminocaproic acid or D-Leu. <400>
SEQUENCE: 27 Lys Lys Tyr Leu Xaa 1 5 <210> SEQ ID NO: 28
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism:Artificial <220> FEATURE:
<223> OTHER INFORMATION: The C-terminal residue is amidated;
residue 5 is aminocaproic acid or D-Leu. <400> SEQUENCE: 28
Lys Lys Tyr Leu Xaa 1 5 <210> SEQ ID NO: 29 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Unknown
<220> FEATURE: <223> OTHER INFORMATION: Description of
Unknown Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The C-terminal residue is amidated; residues 5 and 6
are aminocaproic acid or D-Leu. <400> SEQUENCE: 29 Lys Lys
Tyr Leu Xaa Xaa 1 5 <210> SEQ ID NO: 30 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The C-terminal residue is amidated; residue 5 is
aminolauric acid or D-Lys. <400> SEQUENCE: 30 Lys Lys Tyr Leu
Xaa 1 5 <210> SEQ ID NO: 31 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:
<223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The C-terminal residue is amidated; residues 5-7 are
aminocaproic acid or D-Leu. <400> SEQUENCE: 31 Lys Lys Tyr
Leu Xaa Xaa Xaa 1 5 <210> SEQ ID NO: 32 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a lauroyl moiety; The
C-terminal residue is amidated; residue 6 is aminocaproic acid or
D-Leu. <400> SEQUENCE: 32 Asn Ser Ile Leu Asn Xaa 1 5
<210> SEQ ID NO: 33 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The C-terminal
residue is amidated; residue 6 is aminocaproic acid or D-Leu.
<400> SEQUENCE: 33 Asn Ser Ile Leu Asn Xaa 1 5 <210>
SEQ ID NO: 34 <211> LENGTH: 7 <212> TYPE: PRT
<213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The C-terminal
residue is amidated; residues 6 and 7 are aminocaproic acid or
D-Leu. <400> SEQUENCE: 34 Asn Ser Ile Leu Asn Xaa Xaa 1 5
<210> SEQ ID NO: 35 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The N-terminus
is modified with a caproyl moiety; the C-terminal residue is
amidated; residues 6 and 7 are aminocaproic acid or D-Leu.
<400> SEQUENCE: 35 Asn Ser Ile Leu Asn Xaa Xaa 1 5
<210> SEQ ID NO: 36 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The C-terminal
residue is amidated; residues 6-8 are aminocaproic acid or D-Leu.
<400> SEQUENCE: 36 Asn Ser Ile Leu Asn Xaa Xaa Xaa 1 5
<210> SEQ ID NO: 37 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The C-terminal
residue is amidated; residue 6 is aminolauric acid or D-Lys.
<400> SEQUENCE: 37 Asn Ser Ile Leu Asn Xaa 1 5 <210>
SEQ ID NO: 38 <211> LENGTH: 10 <212> TYPE: PRT
<213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The C-terminal
residue is amidated; residue 5 is aminocaproic acid or D-Leu.
<400> SEQUENCE: 38 Lys Lys Tyr Leu Xaa Asn Ser Ile Leu Asn 1
5 10 <210> SEQ ID NO: 39 <211> LENGTH: 11 <212>
TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:
<223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The C-terminal residue is amidated; residues 5 and 6
are aminocaproic acid or D-Leu. <400> SEQUENCE: 39 Lys Lys
Tyr Leu Xaa Xaa Asn Ser Ile Leu Asn 1 5 10 <210> SEQ ID NO:
40 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Unknown <220> FEATURE: <223> OTHER
INFORMATION: Description of Unknown Organism:Artificial <220>
FEATURE: <223> OTHER INFORMATION: The C-terminal residue is
amidated; residues 5-7 are aminocaproic acid or D-Leu. <400>
SEQUENCE: 40 Lys Lys Tyr Leu Xaa Xaa Xaa Asn Ser Ile Leu Asn 1 5 10
<210> SEQ ID NO: 41 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The C-terminal
residue is amidated; residue 5 is aminolauric acid or D-Lys.
<400> SEQUENCE: 41 Lys Lys Tyr Leu Xaa Asn Ser Ile Leu Asn 1
5 10 <210> SEQ ID NO: 42 <211> LENGTH: 12 <212>
TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:
<223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated; residues 5-7 are Ala, D-Ala or
p-amino Phe. <400> SEQUENCE: 42 Lys Lys Tyr Leu Xaa Xaa Xaa
Asn Ser Ile Leu Asn 1 5 10 <210> SEQ ID NO: 43 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Unknown
<220> FEATURE: <223> OTHER INFORMATION: Description of
Unknown Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated. <400> SEQUENCE: 43 Lys Lys
Leu Tyr Ala Ala Ala Asn Ser Ile Leu Asn 1 5 10 <210> SEQ ID
NO: 44 <211> LENGTH: 10 <212> TYPE: PRT <213>
ORGANISM: Unknown <220> FEATURE: <223> OTHER
INFORMATION: Description of Unknown Organism:Artificial <220>
FEATURE: <223> OTHER INFORMATION: The N-terminus is modified
with a stearoyl moiety; the C-terminal residue is amidated.
<400> SEQUENCE: 44 Lys Lys Tyr Leu Pro Asn Ser Ile Leu Asn 1
5 10 <210> SEQ ID NO: 45 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:
<223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated. <400> SEQUENCE: 45 Lys Tyr
Leu Asn Ser Ile Leu Asn
1 5 <210> SEQ ID NO: 46 <211> LENGTH: 9 <212>
TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:
<223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated. <400> SEQUENCE: 46 Lys Lys
Tyr Leu Asn Ser Ile Leu Asn 1 5 <210> SEQ ID NO: 47
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism:Artificial <220> FEATURE:
<223> OTHER INFORMATION: The N-terminus is modified with a
lauroyl moiety; the C-terminal residue is amidated. <400>
SEQUENCE: 47 Lys Lys Tyr Leu Asn Ser Ile Leu Asn 1 5 <210>
SEQ ID NO: 48 <211> LENGTH: 4 <212> TYPE: PRT
<213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The N-terminus
is modified with a stearoyl moiety; the C-terminal residue is
amidated. <400> SEQUENCE: 48 Lys Tyr Leu Asn 1 <210>
SEQ ID NO: 49 <211> LENGTH: 5 <212> TYPE: PRT
<213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The N-terminus
is modified with a stearoyl moiety; the C-terminal residue is
amidated; residue 5 is D-Ala or D-Tyr. <400> SEQUENCE: 49 Lys
Lys Tyr Leu Xaa 1 5 <210> SEQ ID NO: 50 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated. <400> SEQUENCE: 50 Lys Lys
Tyr Leu Leu 1 5 <210> SEQ ID NO: 51 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated; residue 2 is aminolauric acid or
D-Lys. <400> SEQUENCE: 51 Lys Xaa Tyr Leu 1 <210> SEQ
ID NO: 52 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Unknown <220> FEATURE: <223> OTHER
INFORMATION: Description of Unknown Organism:Artificial <220>
FEATURE: <223> OTHER INFORMATION: The N-terminus is modified
with a stearoyl moiety; the C-terminal residue is amidated; residue
1 is aminolauric acid or D-Lys. <400> SEQUENCE: 52 Xaa Lys
Tyr Leu 1 <210> SEQ ID NO: 53 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated; residues 1 and 2 are aminolauric
acid or D-Lys. <400> SEQUENCE: 53 Xaa Xaa Tyr Leu 1
<210> SEQ ID NO: 54 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The N-terminus
is modified with a stearoyl moiety; the C-terminal residue is
amidated. <400> SEQUENCE: 54 Lys Lys Phe Leu 1 <210>
SEQ ID NO: 55 <211> LENGTH: 4 <212> TYPE: PRT
<213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The N-terminus
is modified with a stearoyl moiety; the C-terminal residue is
amidated; residue 4 is aminocaproic acid or D-Leu. <400>
SEQUENCE: 55 Lys Lys Tyr Xaa 1 <210> SEQ ID NO: 56
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism:Artificial <220> FEATURE:
<223> OTHER INFORMATION: The N-terminus is modified with a
stearoyl moiety; the C-terminal residue is amidated. <400>
SEQUENCE: 56 Lys Lys Trp Leu 1 <210> SEQ ID NO: 57
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism:Artificial <220> FEATURE:
<223> OTHER INFORMATION: The N-terminus is modified with a
stearoyl moiety; the C-terminal residue is amidated; residue 3 is
Ala, D-Ala or p-amino Phe. <400> SEQUENCE: 57 Lys Lys Xaa Leu
1 <210> SEQ ID NO: 58 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The N-terminus
is modified with a stearoyl moiety; the C-terminal residue is
amidated; residue 2 is Orn. <400> SEQUENCE: 58 Lys Xaa Tyr
Leu 1 <210> SEQ ID NO: 59 <211> LENGTH: 4 <212>
TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:
<223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated; residue 1 is Orn. <400>
SEQUENCE: 59 Xaa Lys Tyr Leu 1 <210> SEQ ID NO: 60
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism:Artificial <220> FEATURE:
<223> OTHER INFORMATION: The N-terminus is modified with a
stearoyl moiety; the C-terminal residue is amidated; residues 1 and
2 are Orn. <400> SEQUENCE: 60 Xaa Xaa Tyr Leu 1 <210>
SEQ ID NO: 61 <211> LENGTH: 4 <212> TYPE: PRT
<213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The N-terminus
is modified with a oleic acid moiety; the C-terminal residue is
amidated. <400> SEQUENCE: 61 Lys Lys Tyr Leu 1 <210>
SEQ ID NO: 62 <211> LENGTH: 4 <212> TYPE: PRT
<213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The N-terminus
is modified with a propyl moiety; the C-terminal residue is
amidated. <400> SEQUENCE: 62 Lys Lys Tyr Leu 1 <210>
SEQ ID NO: 63 <211> LENGTH: 10 <212> TYPE: PRT
<213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The N-terminus
is modified with a stearoyl moiety; the C-terminal residue is
amidated. <400> SEQUENCE: 63 Lys Lys Tyr Leu Ala Ala Lys Lys
Tyr Leu 1 5 10 <210> SEQ ID NO: 64 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: The N-terminus is modified with a stearoyl moiety; the
C-terminal residue is amidated. <400> SEQUENCE: 64 Lys Lys
Tyr Leu Pro Pro Lys Lys Tyr Leu 1 5 10 <210> SEQ ID NO: 65
<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism:Artificial <220> FEATURE:
<223> OTHER INFORMATION: The N-terminus is modified with a
stearoyl moiety; the C-terminal residue is amidated; residue 5 is
aminocaproic acid. <400> SEQUENCE: 65 Lys Lys Tyr Leu Xaa Lys
Lys Tyr Leu 1 5
<210> SEQ ID NO: 66 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown Organism:Artificial
<220> FEATURE: <223> OTHER INFORMATION: The N-terminus
is modified with a stearoyl moiety; the C-terminal residue is
amidated; residue 5 is aminolauric acid. <400> SEQUENCE: 66
Lys Lys Tyr Leu Xaa Lys Lys Tyr Leu 1 5 <210> SEQ ID NO: 67
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism:Artificial <220> FEATURE:
<223> OTHER INFORMATION: The N-terminus is modified with a
stearoyl moiety; the C-terminal residue is amidated. <400>
SEQUENCE: 67 Lys Lys Tyr Leu Leu 1 5 <210> SEQ ID NO: 68
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism:Artificial <220> FEATURE:
<223> OTHER INFORMATION: The N-terminus is modified with a
caproyl moiety; the C-terminal residue is amidated. <400>
SEQUENCE: 68 Asn Ser Ile Leu Asn 1 5 <210> SEQ ID NO: 69
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism:Artificial <220> FEATURE:
<223> OTHER INFORMATION: The N-terminus is modified with a
lauroyl moiety; the C-terminal residue is amidated. <400>
SEQUENCE: 69 Asn Ser Ile Leu Asn 1 5 <210> SEQ ID NO: 70
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism:Artificial <220> FEATURE:
<223> OTHER INFORMATION: The N-terminus is modified with a
stearoyl moiety; the C-terminal residue is amidated. <400>
SEQUENCE: 70 Lys Lys Lys Tyr Leu Asp 1 5 <210> SEQ ID NO: 71
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism:Artificial <220> FEATURE:
<223> OTHER INFORMATION: The N-terminus is modified with a
stearoyl moiety; the C-terminal residue is amidated. <400>
SEQUENCE: 71 Cys Lys Lys Tyr Leu Cys 1 5 <210> SEQ ID NO: 72
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism:Artificial <220> FEATURE:
<223> OTHER INFORMATION: The N-terminus is modified with a
stearoyl moiety; the C-terminal residue is amidated. <400>
SEQUENCE: 72 Cys Lys Lys Tyr Leu Lys 1 5 <210> SEQ ID NO: 73
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism:Artificial <220> FEATURE:
<223> OTHER INFORMATION: The N-terminus is modified with a
stearoyl moiety; the C-terminal residue is amidated. <400>
SEQUENCE: 73 Leu Asn Ser Ile Leu Asn 1 5 <210> SEQ ID NO: 74
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism:Artificial <220> FEATURE:
<223> OTHER INFORMATION: The N-terminus is modified with a
stearoyl moiety; the C-terminal residue is amidated. <400>
SEQUENCE: 74 Asn Ser Ile Tyr Leu Asn 1 5 <210> SEQ ID NO: 75
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown Organism:Artificial <220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 1 is a residue of a
hydrophobic aliphatic amino acid <400> SEQUENCE: 75 Xaa Asn
Ser Ile Leu Asn 1 5 <210> SEQ ID NO: 76 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: Xaa at position 1 is a residue of a hydrophobic
aliphatic amino acid <400> SEQUENCE: 76 Xaa Asn Ser Tyr Leu
Asn 1 5 <210> SEQ ID NO: 77 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:
<223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: Xaa at position 6 is a residue of a hydrophobic
aliphatic amino acid <400> SEQUENCE: 77 Val Ala Lys Lys Tyr
Xaa Asn 1 5 <210> SEQ ID NO: 78 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: Xaa at position 6 is a residue of a hydrophobic
aliphatic amino acid <400> SEQUENCE: 78 Ala Val Lys Lys Tyr
Xaa Asn 1 5 <210> SEQ ID NO: 79 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown
Organism:Artificial <220> FEATURE: <223> OTHER
INFORMATION: Any one or all of residues 1-7 may be present or
absent; residue 1 is Val, Ala, or absent; residue 2 is Val, Ala, or
absent; residue 3 is Lys, Val, or absent; residue 4 is Lys or
absent; residue 5 is Tyr, Lys, or absent; residue 6 is a
hydrophobic aliphatic amino acid residue, Tyr, Lys, or absent;
residue 7 is Asn, a hydrophobic aliphatic amino acid residue, or
absent; residue 9 is Ile or Tyr; the C-terminal residue is amidated
or modified with a lipophilic moiety. <400> SEQUENCE: 79 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Leu Asn 1 5 10 <210> SEQ ID
NO: 80 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Unknown <220> FEATURE: <223> OTHER
INFORMATION: Description of Unknown Organism:Artificial <220>
FEATURE: <223> OTHER INFORMATION: Any one or all of residues
1, 2 and 3 may be present or absent; residue 1 is Ala, Val, or
absent; residue 2 is Ala, Val, or absent; residue 3 is Ala, Val,
Lys, D-Lys, Orn or absent; one of residues 1, 2, 3 or 4 is the
N-terminal residue, and may be modified with a lipophilic moiety;
residue 4 is Lys, D-Lys, or Orn; residue 5 is Tyr, D-Tyr, Phe, Trp,
or p-amino Phe; residue 6 is a hydrophobic amino acid residue; any
one or all of residues 7-11 may be present or absent; residue 7 is
Asn, Ser, Ile, Tyr, Leu, Nle, D-Ala, or absent; residue 8 is Ser or
absent; residue 9 is Ile, Tyr, or absent; residue 10 is Leu or
absent; residue 11 is Asn or absent; the C-terminal residue is
amidated or modified with a lipophilic moiety. <400>
SEQUENCE: 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10
* * * * *